Regulation of cancer using natural compounds and/or diet

ABSTRACT

The current invention is directed to a treatment of a proliferative disease comprising administering to a subject in need of such treatment, a composition comprising epigallocatechin-3-gallate (EGCG), curcumin, glucosinolates and, optionally Daikon radish sprout, alone or in combination with providing a ketogenic diet or a modified ketogenic diet to the subject. The invention also provides a composition comprising medium chain triglycerides, epigallocatechin-3-gallate, curcumin, compositions comprising glucosinolates and/or derivatives thereof, such as glucoraphanin and its breakdown product sulforaphane, (SFN) (which are found at high levels in broccoli sprouts or sprouts of other cruciferous vegetables), and, optionally Daikon radish sprout.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 61/784,386, filed Mar. 14, 2013, the disclosure of which is herebyincorporated by reference in its entirety, including all figures, tablesand amino acid or nucleic acid sequences.

Due to a shift in most cancer cells from oxidative phosphorylation toaerobic glycolysis (known as the Warburg effect) cancer can be viewed asa metabolic disease where energy flux is shifted from a highly efficientmethod of generating energy (36 molecules of ATP from 1 molecule ofglucose) to an inefficient method (4 molecules of ATP from one moleculeof glucose). The result is that cancer cells expend an enormous amountof glucose to survive and multiply. While controversy exists as to therelationship between the Warburg effect and altered signaling pathways,the combined reliance of tumor cells for excessive amounts of glucoseand signaling pathway alterations suggest that targeting these tworelated phenomena may provide better outcomes in cancer treatment.

Most cancer treatments employ the use of toxic chemicals aimed atkilling cancerous cells. While these treatments are highly effective,unfortunately, they have similar effects on normal, non-cancerous cellsas well. The key to developing an effective and well-toleratedchemotherapy regime is to balance the positive tumor killing effects ofthe compounds with the toxic side effects. The use of non-toxiccompounds that are able to target altered signaling pathways andinfluence energy flux may provide an effective and tolerable treatment.An additional advantage of using non-toxic approach is the ability toapply multiple agents simultaneously with reduced chances of cumulativetoxicity. The current invention provides treatment of proliferativedisorders that target altered signaling pathways and energy flux incancerous cells.

BRIEF SUMMARY OF THE INVENTION

The current invention provides a treatment of a proliferative diseasecomprising, administering to a subject in need of a treatment against aproliferative disease, a composition comprising one or more naturalproducts (compounds) and, optionally, simultaneously providing to thesubject a low carbohydrate diet. In certain embodiments of theinvention, the subject consumes (or is provided) a modified ketogenicdiet (mKD) or a ketogenic diet (KD). Thus, the current invention alsoprovides a therapy for a subject in need of a treatment against aproliferative disorder, the therapy comprising administering to asubject consuming a mKD or KD diet a composition comprising one or morenatural compounds (component(s)) selected from,epigallocatechin-3-gallate (EGCG), curcumin, compositions comprisingglucosinolates and/or derivatives thereof, such as glucoraphanin and/orsulforaphane (SFN) (as found in broccoli sprouts or sprouts of othercruciferous vegetables), and, optionally, Daikon radish sprout, a Daikonradish sprout extract or a powder of said extract or the Daikon radishsprout. In another aspect of the invention, the method of treating aproliferative disorder comprises administering one or more component(s)selected from, epigallocatechin-3-gallate (EGCG), curcumin, compositionscomprising glucosinolates and/or derivatives thereof, such asglucoraphanin and/or SFN (derived from sources such as broccoli sprouts,sprouts of other cruciferous vegetables or cruciferous vegetablesthemselves) and, optionally, Daikon radish sprout, a Daikon radishsprout extract or a powder of said extract or the Daikon radish sprout,and, optionally, simultaneously providing a low carbohydrate, mKD or KDdiet.

Another aspect of the invention provides methods that attenuate/reducingthe loss or the proliferative ability of neural stem cells (NSC) ortheir progeny [collectively called precursor cells] of the CNS in asubject developing a tumor or having a tumor or in a subject having aneurodegenerative disease or disorder, such as Parkinson's disease (PD),Alzheimer's disease (AD), stroke, Amyotrophic lateral sclerosis (ALS),Acute disseminated encephalomyelitis (ADEM) and Neuromyelitis optica(NMO) or that which is associated to aging or age-related cognitivedecline. Thus, various embodiments of this aspect of the inventionprovide methods of attenuating/reducing the loss in activity ofprecursor cells or a loss in the number of precursor cells in the CNS ina subject developing a tumor or having a tumor or in a subject having aneurodegenerative disease or disorder or a age related reduction in CNSfunction, comprising administering to a subject a composition comprisingone or more natural compounds (component(s)) selected from,epigallocatechin-3-gallate (EGCG), curcumin, compositions comprisingglucosinolates and/or derivatives thereof, such as glucoraphanin and/orsulforaphane (SFN) (such as broccoli sprouts, sprouts of othercruciferous vegetables or cruciferous vegetables themselves), and,optionally, Daikon radish sprout, a Dailon radish sprout extract or apowder of said extract or the Daikon radish sprout and, optionally,simultaneously providing a low carbohydrate, mKD or KD diet.

The current invention also provides a composition comprising one or moreof the following natural compounds (components): EGCG, curcumin,compositions comprising glucosinolates and/or derivatives thereof, suchas SFN and/or glucoraphanin (optionally in the form of broccoli sprouts,the sprouts of other cruciferous vegetables or cruciferous vegetablesthemselves), and Daikon radish sprout, a Dailon radish sprout extract ora powder of said extract or the Daikon radish sprout and, optionally,medium chain triglycerides (MCT).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Effect of mKD on blood glucose levels. Blood glucose level wascompared between animals that were fed respectively for 2 weeks with thedifferent diets [control, ketogenic diet (KD), modified ketogenic diet(mKD) or mKD/NP]. Glucose level was similar between the KD, mKD, NaturalProducts (NP) and mKD/NP groups, which were significantly decreasedcompared to control. *, ***, compared to control, p<0.01, 0.001, 1-wayANOVA. Treatments composition is as follow: Control (55% carbohydrate,30% protein, 15% fat), KD (92% Fat, 3% carbohydrate, 5% protein),mKD=10% carbohydrate, 60% Fat (half coming from MCT, Neobee 598), 30%Protein, Natural Products (NP) [55% carbohydrate, 30% protein, 15%fat+SFN (25 mg/kg; BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200mg/kg)], mKD/NP=mKD+Natural Products (NP).

FIG. 2: Effect of mKD on blood ketone levels. Blood ketones levels wascompared between animals that were fed respectively for 2 weeks with thedifferent diets [control, ketogenic diet (KD), modified ketogenic diet(mKD) or mKD/NP]. Ketones level was similar between the KD, mKD, NaturalProducts (NP) and mKD/NP groups, which were significantly decreasedcompared to control. ***, compared to control, p<0.001, 1-way ANOVA.Treatments composition is as follow: Control (55% carbohydrate, 30%protein, 15% fat), KD (92% Fat, 3% carbohydrate, 5% protein), mKD=10%carbohydrate, 60% Fat (half coming from MCT, Neobee 598), 30% Protein,Natural Products (NP) [55% carbohydrate, 30% protein, 15% fat+SFN (25mg/kg; BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg)],mKD/NP=mKD+Natural Products (NP).

FIG. 3: Effect of mKD/NP on body weight. Toxicity of mKD/NP was assessedby monitoring body weight over 16 days. Over the course of the study themKD/NP treated animals did not lose weight and even shown and increasedbody weight compared to control animals. p<0.005, Linear regression.

FIG. 4: Toxicology—Blood tests. Toxicity was assessed after 4 weeks oftreatment (mKD/NP) via plasma measurements of the following analytes(Comparative Clinical Pathology Services, LLC): creatinine (kidney),alanine transaminase (ALT, liver), aspartate aminotransferase (AST,liver), and alkaline phosphatase (ALP, pancreas). No difference wasobserved between mKD/NP treated animals compared to controls. p>0.1,1-sample t-test.

FIG. 5: Non-Tumor Death—TMZ vs. mKD/NP. Mortality rate unrelated totumor was monitored during the course of treatments. Compared toconventional therapy (Temozolomide [TMZ], 20 mg/kg), mKD/NP treatmentdecreased mortality by 10 fold. **, p<0.01, t-test.

FIG. 6: Body weight—TMZ vs. mKD/NP. Toxicity/safety of mKD/NP wascompared to standard of care (TMZ, 20 mg/kg) by monitoring body weightafter 4 days of treatment. No difference was observed between controland mKD/NP treated animals (p>0.05, 1-way ANOVA) whereas animals treatedwith conventional treatment showed significant loss of body weightcompared to control and mKD/NP. ##, ###, p<0.001, p<0.0001, 1-way ANOVA,compared to TMZ.

FIG. 7: NP in vitro—Fold Expansion. Primary human GBM stem cell lineswere treated daily for 4 out of 5 days in culture with EGCG (8 μM),Curcumin (0.5 μM) and sulforaphane (2.5 μM) or a combination of allthree NPs (E+C+S). Cells were passed after 5-7 days and cell counts wereperformed. All individual compounds exhibited a significant reduction inmean daily fold expansion of GB cells. The combination of the threenatural products (NP) together demonstrated the strongest effect. Thesynergistic effect of the combination suggests that each component of NPis affecting non-overlapping mechanisms. *, **, ***, p<0.01, p<0.01,p<0.0001, 1-way ANOVA, compared to control. ###, p<0.001, 1-way ANOVA,compared to NP. Micrographs show cultures after 4 days of exposure tothe different treatments.

FIG. 8: Kaplan-Meier (KM) survival curves—Individual NPs vs.Combination. NOD/SCID animals were inoculated with 1M hGB cells in theright flank. Tumor progression was followed using calipers by recording2 measurements of tumor diameter and converting this into a volume usingthe following formula: (4/3)πR³. For spheroid tumors the twomeasurements were averaged to determine the diameter of the sphere. Inthe case of ellipsoid tumors (i.e. prolate or oblate spheroid mass) theformula used was: (4/3)π*(d/2)*(d/2)². In this case the secondmeasurement “d²” would count twice and “d” only once. For prolatespheroids, the long measurement occurs once while the short measurementoccurs twice. Conversely, for the oblate spheroid tumors, the longmeasurement occurs twice while the short one occurs only once. Followingthis criteria, tumor volume was tracked over time. Treatments wereinitiated when a palpable mass was identified [approximately 65 mm³].Animals were sacrificed when they reached endpoint (tumor volume of 1700mm³). The fraction of animals living as a function of time isrepresented using Kaplan-Meier survival curves. Animals treated with NPdemonstrated a significant improvement over controls or animals treatedwith individual component (*, **, p<0.05, p<0.005, Log rank test).

FIGS. 9A-9B: NP—Effect on cancer stem cells (CSCs). Patient derived GBcell lines cultured in defined medium were treated with NP. After 5-7days in culture, spheres were harvested, dissociated into single cellssuspension and plated at low density in 96 well plates in controlconditions. Seven to ten days later the number of spheres was counted(A: clonogenic frequency) and sized (B). This assay is able to accessthe effects of treatment on the sphere forming cells (i.e clonogenicfrequency in A) and on the proliferative potential of each of the clones(B). Exposure of human GB cells to NPs for 7 days in vitro results in asignificant reduction in the clonogenic frequency and the proliferativeability of the clones. **, p<0.01, t-test.

FIGS. 10A-10B: NP—Effect CSCs. NP targets tumor-propagating cell invitro. A) Patient derived hGB cells were serially passed in culture for5 passages under a variety of treatment conditions. Cells were treatedwith EGCG (8 μM), curcumin (0.5 μM, SFN (2.5 μM) or their combination(NP). NP had the greatest growth inhibitory effect. *, *** p<0.05,p<0.001 compared to control, ###p<0.001 compared to NP, Linearregression. B) The rate of tumor propagating cell aka cancer stem cell(CSC) expansion (Kll) is directly correlated to the probability CSCsundergo self-renewing symmetric division and can be calculated by takingthe natural logarithm of the fold expansion and dividing by the passagetime (Deleyrolle et al., 2011). Human GB cells were cultured in theneurosphere assay over 5 passages during which CSC expansion rate wasevaluated. Only SFN or NP treated groups demonstrated significantdecrease of CSCs self-renewing symmetric division rate compared tocontrol. Also NP exhibited the greatest effect suggesting a uniquesynergistic effect. ***, p<0.001 compared to control, ^(###), p<0.001compared to NP, 1-way ANOVA.

FIG. 11: Daily fold expansion—Effect of NP and TMZ in vitro. Primaryhuman GB stem cell lines were treated daily for 4 out of 5 days inculture TMZ alone or in combination with EGCG (8 μM), Curcumin (0.5 μM),sulforaphane (2.5 μM) or a combination of all three (NP). Cells werepassed after 5-7 days and cell counts performed. The addition of EGCG,curcumin or SFN to TMZ treatment resulted in a significant reduction inthe mean daily fold expansion of hGB cells compared to control. However,NP had the greatest effect demonstrating synergistic effect. The stars(*) are compared to control, # to TMZ alone and $ to the differentcombinations of TMZ with each of the individual natural products. 1symbol, p<0.05, 3 symbols, p<0.001, 1-way ANOVA.

FIG. 12: Effect of mKD on body weight—24 day treatment. Toxicity andnutritional sufficiency of mKD were assessed by monitoring body weight.Animals fed with a KD demonstrated a significant weight loss compared tocontrols whereas mKD treated group did not show weight loss over thecourse of 24 days of treatment and exhibited a body weight similar tocontrols. *, **, p<0.05, p<0.005, 1-way ANOVA.

FIG. 13: Effect of mKD on tumor volume progression. NOD/SCID animalswere inoculated with 1M hGB cells in the right flank. Tumor dimensionswere monitored 3 times per week using calipers and volume wascalculated. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. Animals were sacrificed when theyreached endpoint (1700 mm³). Animals treated with KD or mKD showedsimilar tumor progression and demonstrated a significant slowerprogression compared to controls (**, p<0.005, two-way ANOVA).

FIG. 14: Effect of mKD on KM curve. NOD/SCID animals were inoculatedwith 1M hGB cells in the right flank. Tumor dimensions were monitored 3times per week using calipers and volume was calculated. Treatments wereinitiated when a palpable mass was identified [approximately 65 mm³].Animals were sacrificed when they reached endpoint based on a tumorvolume of 1700 mm³ calculated from measurements made with a caliper. Thefraction of animals living as a function of time is represented usingKaplan-Meier survival curves. Animals treated with KD or mKD showedsimilar survival and demonstrated a significant improvement overcontrols (**, p<0.005, Log rank test).

FIG. 15: Effect of mKD progression free survival. NOD/SCID animals wereinoculated with 1M hGB cells in the right flank. Tumor volume wascalculated 3 times per week and the time from a barely palpable tumor[approximately 65 mm³] to a tumor of a significant size [300 mm³] wascalculated. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. Animals treated with KD or mKD showedsimilar progression free survival (time during which tumor volume ismaintained lower than 300 mm³) and demonstrated a significantimprovement over controls (*, **, p<0.05, p<0.005, t-test).

FIG. 16: Effect of mKD on overall survival. NOD/SCID animals wereinoculated with 1M hGB cells in the right flank. Tumor dimensions weremonitored 3 times per week using calipers and volume was calculated.Treatments were initiated when a palpable mass was identified[approximately 65 mm³]. Animals were sacrificed when they reachedendpoint (1700 mm³). The average time to reach endpoint volume was thencompared. Animals treated with KD or mKD showed similar overall survivaland demonstrated a significant improvement over controls (**, p<0.005,Log rank test).

FIG. 17: Effect of mKD/NP on proliferation (In vitro). Human GB cellswere plated at 50,000 cells per ml in the neurosphere assay. The cellswere treated with the indicated treatments and harvested for cell numberquantification after 7 days of culture. The three treatment groupsdemonstrated significant decrease of proliferation compared to control,with mKD/NP treated animals showing significant difference compared tomKD and NP groups. **, compared to control, ## compared to mKD/NP,p<0.001, t-test. Details of the treatments: [1] mKD=4 mM ketones [BetaHydroxybutyrate](Single treatment applied 2 days post plating), [2]NP=EGCG [8 μM]+SFN [2.5 μM]+Curcumin [0.5 μM]: daily treatment from day3 to day 6, [3] mKD/NP=4 mM ketones+EGCG [8 μM]+SFN [5 μM]+Curcumin [0.5μM]. Of note, glucose level was 65 mg/dL in the mKD and mKD/NP groups,and 130 mg/dL in the control and NP groups.

FIG. 18: Effect of mKD/NP on CSC. Sphere forming frequency was measuredto evaluate the effect of the different treatments on the proliferationof CSCs. Human GB cells were plated at 50,000 cells per ml in theneurosphere assay. The cells were treated with the indicated treatmentsand harvested to be plated in regular medium (no treatment) at clonaldensity for comparison of their respective sphere formation ability.mKD/NP treated cells showed significant decrease of sphere formingability compared to the control group. **, p<0.005, t-test.

FIG. 19: Effect of mKD/NP on CSC expansion. The rate of cancer stem cell(CSC) expansion (Kll) is directly correlated to the probability CSCsundergo self-renewing symmetric division and can be calculated by takingthe natural logarithm of the fold expansion and dividing by the passagetime (Deleyrolle et al., 2011). Human GB cells were cultured in theneurosphere assay over 4 passages during which CSC expansion rate wasevaluated. The three treatment groups demonstrated significant decreaseof CSCs self-renewing symmetric division rate compared to control.mKD/NP group also demonstrated significant decrease compared to mKD andNP groups. *, **, ***, p<0.05, p<0.01, p<0.001, compared to mKD/NP,t-test.

FIG. 20: Effect of mKD/NP on Tumor Progression. NOD/SCID animals wereinoculated with 1M hGB cells in the right flank. Tumor dimensions weremonitored 3 times per week using calipers and volume was calculated.Treatments were initiated when a palpable mass was identified[approximately 65 mm³]. Animals were sacrificed when they reachedendpoint (1500 mm³). Animals treated with mKD/NP demonstrated asignificant slower tumor progression compared to controls or animalstreated with mKD or NP (**, *** p<0.01, p<0.002, two-way ANOVA). Theseresults demonstrate a synergistic effect in vivo between mKD and NP.

FIG. 21: Effect of mKD/NP on KM curve. NOD/SCID animals were inoculatedwith 1M hGB cells in the right flank. Tumor volume was monitored 3 timesper week. Treatments were initiated when a palpable mass was identified[approximately 65 mm³]. Animals were sacrificed when they reachedendpoint (1500 mm³). The fraction of animals living as a function oftime is represented using Kaplan-Meier survival curves. Animals treatedwith mKD/NP demonstrated a significant improvement over controls oranimals treated with mKD or NP (**, *** p<0.01, p<0.002, Log rank test).

FIG. 22: Effect of mKD/NP on overall survival. NOD/SCID animals wereinoculated with 1M hGB cells in the right flank. Tumor volume wasmonitored 3 times per week. Treatments were initiated when a palpablemass was identified [approximately 65 mm³]. Animals were sacrificed whenthey reached endpoint (1500 mm³). The average time to reach endpointvolume was then compared. Animals treated with mKD/NP demonstrated asignificant increase of overall survival compared to controls or animalstreated with mKD or NP (*, **, *** p<0.05, p<0.01, p<0.002, compared tomKD/NP, t-test).

FIG. 23: Effect of mKD/NP on Progression Free Survival. NOD/SCID animalswere inoculated with 1M hGB cells in the right flank. Tumor volume wascalculated 3 times per week and the time from a barely palpable tumor[approximately 65 mm³] to a tumor of a significant size [300 mm³] wascalculated. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. mKD/NP animals demonstrated asignificant increase of tumor progression free survival (time duringwhich tumor volume is maintained lower than 300 mm³) compared tocontrols or animals treated with mKD or NP (***, p<0.0001, compared tomKD/NP, 1-way ANOVA). These results suggest a synergistic effect betweenmKD and NP treatments.

FIG. 24: Effect of mKD/NP on KM after intracranial inoculation of hGBcells. NOD/SCID animals were inoculated with 200K hGB cells in thestriatum. Treatments were initiated 3 days post implant. Animals weresacrificed when they reached endpoint marked by the development ofneurologic signs (including, but not limited to, lethargy, paralysis, orseizure). The fraction of animals living as a function of time isrepresented using Kaplan-Meier survival curves. mKD/NP treated animalsdemonstrated a significant increased survival compared to controlanimals (*, p=0.014, Log-Rank test).

FIG. 25: Effect of mKD/NP on overall survival after intracranialinoculation of hGB cells. NOD/SCID animals were inoculated with 200K hGBcells in the striatum. Treatments were initiated 3 days post implant.Animals were sacrificed when they reached endpoint marked by thedevelopment of neurologic signs (including, but not limited to,lethargy, paralysis, or seizure). The average time to reach endpointvolume (i.e. overall survival time) was then compared. mKD/NP treatedanimals demonstrated a significant increased overall survival comparedto controls (*, p<0.05, t-test).

FIG. 26: Tumor Progression—TMZ vs. mKD/NP. NOD/SCID animals wereinoculated with 1M hGB cells in the right flank. Tumor volume wasmonitored 3 times per week using a caliper and tumor volume wascalculated. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. Animals were sacrificed when theyreached endpoint (1500 mm³). Animals treated with standard of care(temozolomide, TMZ, 5 mg/kg) or mKD/NP demonstrated a similar andsignificant slower tumor progression compared to controls (***,p<0.0001, two-way ANOVA).

FIG. 27: Progression Free Survival—TMZ vs. mKD/NP. NOD/SCID animals wereinoculated with 1M hGB cells in the right flank. Tumor volume wascalculated 3 times per week and the time from a barely palpable tumor[approximately 65 mm³] to a tumor of a significant size [300 mm³] wascalculated. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. Animals treated with standard of care(temozolomide, TMZ, 5 mg/kg) or mKD/NP demonstrated a similar andsignificant increase of progression free survival time (time duringwhich tumor volume is maintained lower than 300 mm³) compared tocontrols (* p<0.05, t-test).

FIG. 28: mKD/NP—Adjuvant Tumor Progression. NOD/SCID animals wereinoculated with 1M TMZ sensitive hGB cells in the right flank. Tumorvolume was monitored 3 times per week using a caliper and tumor volumewas calculated. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. Animals treated with standard of care(temozolomide, TMZ, 5 mg/kg) or mKD/NP demonstrated a similar andsignificant slower tumor progression compared to controls. Thecombination of standard of care with mKD/NP showed a significantdecrease of tumor progression compared to controls and mKD/NP treatedgroup. **, ***, p<0.005, p<0.0001, two-way ANOVA.

FIG. 29: Effect of mKD/NP on tumor progression of TMZ resistant cells.NOD/SCID animals were inoculated with 1M TMZ unresponsive hGB cells inthe right flank. Tumor volume was monitored 3 times per week using acaliper and tumor volume was calculated. Treatments were initiated whena palpable mass was identified [approximately 65 mm³]. Animals treatedwith mKD/NP demonstrated a significant slower tumor progression comparedto controls demonstrating the efficacy of this treatment as a secondline therapy after resistance to conventional treatment has beendeveloped. The combination of standard of care (TMZ, 5 mg/kg) withmKD/NP showed a significant decrease of tumor progression compared tocontrol, TMZ and mKD/NP treated groups. This result demonstrates theability of mKD/NP to re-sensitize cells to conventional treatment afteracquired resistance. ***, p<0.0001, two-way ANOVA.

FIG. 30: Effect of mKD/NP on time to tumor initiation—GB. Animals wereplaced on mKD/NP for 2 months prior to sub-Q tumor implantation. After 2months of treatment the NOD/SCID animals were inoculated with 1M hGBcells in the right flank. Tumor growth was then monitored 3 times perweek to determine the time between tumor cell implantation and the timea tumor could be palpable (i.e. reaching a volume approximating 65 mm³).The graph depicts the average time between implant and positivepalpation. The mKD/NP treated group demonstrated a time to tumorinitiation approximately 3 times greater than in the controls (***,p<0.01, t-test).

FIG. 31: Effect of mKD/NP on tumor forming frequency—GB. Animals wereplaced on mKD/NP for 2 months prior to sub-Q tumor implantation. After 2months of treatment the NOD/SCID animals were inoculated with 1M hGBcells in the right flank. Tumor growth was then monitored 3 times perweek to determine the time between tumor cell implantation and the timea tumor could be palpable (i.e. reaching a volume approximating 65 mm³).The percentage of animals that had developed tumor was recorded. mKD/NPpre-treated group showed a 60% decrease in tumor initiation compared tocontrols.

FIG. 32: Effect of mKD/NP on tumor forming frequency—Lung Cancer.NOD/SCID animals were treated for 2 weeks with control diet or mKD/NPbefore to be inoculated with 2M of lung carcinoma cells (A549) in theright flank. Tumor growth was then monitored 3 times per week. 21 daysafter implant almost 90% of the control animals developed tumor whereasonly 50% of the animals treated with mKD/NP showed tumor formation.

FIGS. 33A-33B: Effect of mKD/NP on neural stem cell (NSC) activity. A)mKD/NP protects neural stem cells from dysregulation related to tumordevelopment. It has been demonstrated that the presence of a tumor cancreate a chronic inflammatory response sufficient to induce damage andcellular dysregulation in tissues distant from the tumor site (Redon etal., 2010). We demonstrated that development of a tumor mass followingsubcutaneous implant of hGB cells in the right flank of NOD/SCID animalsdownregulated neural stem cell activity (based on BrdU incorporation) inarea related to cognition (e.g. hippocampus). Animals treated withmKD/NP did not demonstrate any decrease in NSC activity compared to thenon-tumor bearing group. These results demonstrate a protective effectof mKD/NP on NSC activity. B) hNSC were plated at 20K cells per 100 ulof medium and cultured in the neurosphere assay for 14 days. Starting 2days post plating, the cells were daily treated with EGCG (8 μM),Curcumin (0.5 μM), sulforaphane (SFN, 2.5 μM) or a combination of allthree NP. After 14 days in culture, MTT assay was performed to measurecell viability. Only the combination of the 3 natural products (NP)exhibited a significant effect compared to control. Cells treated withNP displayed a 70% increase in cell viability compared to controls oreach individual component. ***, p<0.0001, compared to NP, 1-way ANOVA.These data demonstrate that NP treatment increases survival of NSCs.

FIG. 34: Optimization mKD/NP—Tumor progression. The presence of DaikonRadish Sprout Powder (DRSP) enhances the effect of mKD/NP. NOD/SCIDanimals were inoculated with 1M hGB cells in the right flank. Tumorprogression was monitored by measuring tumor volume 3 times per weekusing a caliper. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. Animals treated with mKD/NPcontaining DRSP demonstrated a significant slower tumor progressioncompared to controls or animals treated with mKD/NP not containing DRSP(*, ***, p<0.05, p<0.001, Two-way ANOVA).

Treatments:

Control: 55% carbohydrate, 30% protein, 15% fat.

mKD/NP.001=10% carbohydrate, 60% Fat (half coming from MCT, Neobee 598),30% Protein+Natural Products (NP) containing SFN (25 mg/kg; BSP100%),Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

mKD/NP.002=10% carbohydrate, 60% Fat (half coming from MCT, Neobee 598),30% Protein+Natural Products (NP) containing SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

FIG. 35: Optimization mKD/NP—Progression free survival. NOD/SCID animalswere inoculated with 1M hGB cells in the right flank. Tumor volume wascalculated 3 times per week and the time from a barely palpable tumor[approximately 65 mm³] to a tumor of a significant size [300 mm³] wascalculated. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. Animals treated with mKD/NP.002(containing DRSP) demonstrated a significant increase of tumorprogression free survival time (time during which tumor volume ismaintained lower than 300 mm³) compared to controls or animals treatedwith mKD/NP.001 (not containing DRSP). *, p<0.05, F-test, compared tomKD/NP.002.

FIG. 36: Optimization mKD/NP—overall survival. NOD/SCID animals wereinoculated with 1M hGB cells in the right flank. Tumor volume wasmonitored 3 times per week. Treatments were initiated when a palpablemass was identified [approximately 65 mm³]. Animals were sacrificed whenthey reached endpoint (1500 mm³). The average time to reach endpointvolume (i.e. overall survival time) was then compared. Animals treatedwith mKD/NP.002 (containing DRSP) demonstrated a significant increase ofmean survival compared to controls or animals treated with mKD/NP.001(not containing DRSP). *, p<0.05, F-test, compared to mKD/NP.002.

FIGS. 37A-37B: Optimization mKD/NP—Ki67 and pStat3. NOD/SCID animalswere inoculated with 1M hGB cells in the right flank. Tumor volume wasmonitored 3 times per week. Treatments were initiated when a palpablemass was identified [approximately 65 mm³]. Animals were sacrificed whenthey reached endpoint (1500 mm³) and the tumors were harvested andprepared to quantify tumor cell proliferation using Ki67 and pStat3labeling. (A) The percentage of immunoreactive cells was quantifiedusing flow cytometry. Animals treated with mKD/NP.002 (containing DRSP)demonstrated a significant decrease of proliferation compared tocontrols or animals treated with mKD/NP.001 (not containing DRSP). *,**, p<0.05, p<0.001, t-test, compared to mKD/NP.002. (B) Stat3activation (via phosphorylation) is required for proliferation of cancercells in general and in Glioblastoma (Sherry et al., 2009). TargetingStat3 is thus a potential target for cancer therapy. mKD/NP is able toinhibit the phosphorylation of Stat3 compared to controls. This effectis potentiated with mKD/NP.002 when DRSP is included in the treatment asnoted above.

FIG. 38: Effect of mKD/NP on colon cancer—tumor progression. NOD/SCIDanimals were inoculated with 2M of colorectal adenocarcinoma cells(HT-29) in the right flank. Tumor progression was monitored by measuringtumor volume 3 times per week using a caliper. Treatments were initiatedwhen a palpable mass was identified [approximately 65 mm³]. Animals weresacrificed when they reached endpoint (1000 mm³). Animals treated withmKD/NP demonstrated a significant slower tumor progression compared tocontrols (***, p<0.0001, two-way ANOVA).

FIG. 39: Effect of mKD/NP on colon cancer—tumor volume. NOD/SCID animalswere inoculated with 2M of colorectal adenocarcinoma cells (HT-29) inthe right flank. Tumor progression was monitored by measuring tumorvolume 3 times per week using a caliper. Treatments were initiated whena palpable mass was identified [approximately 65 mm³]. Tumor volume wascompared 30 days post treatment initiation. On average, animals treatedwith mKD/NP demonstrated a significant lower tumor volume compared tocontrols (** p<0.01, t-test).

FIG. 40: Effect of mKD/NP on colon cancer—KM curve. NOD/SCID animalswere inoculated with 2M of colorectal adenocarcinoma cells (HT-29) inthe right flank. Tumor progression was monitored by measuring tumorvolume 3 times per week using a caliper. Treatments were initiated whena palpable mass was identified [approximately 65 mm³]. Animals weresacrificed when they reached endpoint (1000 mm³). The fraction ofanimals living as a function of time is represented using Kaplan-Meiersurvival curves. Animals treated with mKD/NP demonstrated a significantimprovement over controls (** p<0.01, Log rank test).

FIG. 41: Effect of mKD/NP on colon cancer—overall survival. NOD/SCIDanimals were inoculated with 2M of colorectal adenocarcinoma cells(HT-29) in the right flank. Tumor progression was monitored by measuringtumor volume 3 times per week using a caliper. Treatments were initiatedwhen a palpable mass was identified [approximately 65 mm³]. Animals weresacrificed when they reached endpoint (1000 mm³). The average time toreach endpoint volume (i.e. overall survival time) was then compared.Animals treated with mKD/NP demonstrated a significant increase ofoverall survival compared to controls (**, p<0.01, t-test). These datademonstrate that mKD/NP is an effective treatment for colon cancer.

FIG. 42: Effect of NP on colon cancer cells (in vitro). Colorectaladenocarcinoma cells (HT-29) were treated daily with EGCG (8 μM),Curcumin (0.5 μM) and sulforaphane (2.5 μM). Once the control culturesbecame confluent, a cell count was performed. All individual compoundsexhibited a significant reduction in cell number. The combination of thethree natural products together (NP) demonstrated the strongest effect.*, ***, p<0.05, p<0.001, compared to control, ##, ###, p<0.01, p<0.001,compared to NP, 1-way ANOVA.

FIG. 43: Effect of mKD/NP on lung cancer—tumor progression. NOD/SCIDanimals were inoculated with 2M of lung carcinoma cells (A549) in theright flank. Tumor progression was monitored by measuring tumor volume 3times per week using a caliper. Treatments were initiated when apalpable mass was identified [approximately 65 mm³]. Animals weresacrificed when they reached endpoint (1000 mm³). Animals treated withmKD/NP demonstrated a significant slower tumor progression compared tocontrols (*** p<0.0001, two-way ANOVA).

FIG. 44: Effect of mKD/NP on lung cancer—tumor volume. NOD/SCID animalswere inoculated with 2M of lung carcinoma cells (A549) in the rightflank. Tumor volume was measured 3 times per week and treatments wereinitiated when a palpable mass was identified [approximately 65 mm³].Tumor volume was compared 31 days post treatment initiation. Animalstreated with mKD/NP demonstrated a significant lower tumor volumecompared to controls (*, p<0.05, t-test).

FIG. 45: Effect of mKD/NP on lung cancer—tumor progression freesurvival. NOD/SCID animals were inoculated with 2M of lung carcinomacells (A549) in the right flank. Tumor volume was calculated 3 times perweek and the time from a barely palpable tumor [approximately 65 mm³] toa tumor of a significant size [300 mm³] was calculated. Treatments wereinitiated when a palpable mass was identified [approximately 65 mm³].Animals treated with mKD/NP demonstrated a significant increasedprogression free survival time (time during which tumor volume ismaintained lower than 300 mm³) compared to controls (**, p<0.01,t-test).

FIG. 46: Effect of mKD/NP on lung cancer—KM curve. NOD/SCID animals wereinoculated with 2M of lung carcinoma cells (A549) in the right flank.Tumor progression was monitored by measuring tumor volume 3 times perweek using a caliper. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. Animals were sacrificed when theyreached endpoint (1000 mm³). The fraction of animals living as afunction of time is represented using Kaplan-Meier survival curves.Animals treated with mKD/NP demonstrated a significant improvement overcontrols (*, p<0.05, Log rank test).

FIG. 47: Effect of mKD/NP on lung cancer—overall survival. NOD/SCIDanimals were inoculated with 2M of lung carcinoma cells (A549) in theright flank. Tumor volume was monitored 3 times per week using acaliper. Treatments were initiated when a palpable mass was identified[approximately 65 mm³]. Animals were sacrificed when they reachedendpoint (1000 mm³). The average time to reach endpoint volume (i.e.overall survival time) was then compared. Animals treated with mKD/NPdemonstrated a significant increased overall survival compared tocontrols (* p<0.05, t-test). These data demonstrate that mKD/NP is aneffective treatment for lung cancer.

FIG. 48: Effect of NP on lung cancer cells (In vitro). Lung carcinomacells (A549) were treated daily with EGCG (8 μM), Curcumin (0.5 μM) andsulforaphane (2.5 μM). Once the control cultures became confluent a cellcount was performed. Individually EGCG and Curcumin do not show astatistically significant effect in reducing cell numbers while SFNdoes. This effect is potentiated when all three compounds (NP) are usedtogether. *, ***, p<0.05, p<0.001, compared to control, ^(#), ^(##),^(###), p<0.05, p<0.01, p<0.001, compared to NP, 1-way ANOVA.

FIG. 49: Effect of NP on breast cancer cell viability. Human breastcancer cells (ZR751) were treated daily with EGCG (8 μM), Curcumin (0.5μM) or SFN (2.5 μM) individually or in combination (NP). Once thecontrol cultures became confluent, cell viability was measured using MTTassay. Individually, none of the natural product showed statisticallysignificant effect compared to controls while their combination (NP)did. ***, p<0.001, compared to control, 1-way ANOVA.

FIG. 50: Effect of mKD/NP on breast cancer—tumor progression. NOD/SCIDanimals were inoculated with 2M of Breast cancer cells (ZR751) in theright flank. Tumor progression was monitored by measuring tumor volume 3times per week using a caliper. Treatments were initiated when apalpable mass was identified [approximately 65 mm³]. Animals weresacrificed when they reached endpoint (1000 mm3). Animals treated withmKD/NP demonstrated a significant slower tumor progression compared tocontrols (*** p<0.0001, two-way ANOVA).

FIG. 51: Effect of mKD/NP on breast cancer—tumor volume (day 70).NOD/SCID animals were inoculated with 2M of Breast cells (ZR751) in theright flank. Tumor volume was monitored 3 times per week using acaliper. Treatments were initiated when a palpable mass was identified[approximately 65 mm³]. Tumor volume was compared 70 days post treatmentinitiation. Animals treated with mKD/NP demonstrated a significant lowertumor volume compared to controls (**, p<0.01, t-test).

FIG. 52: Effect of mKD/NP on breast cancer—tumor volume (day 145).NOD/SCID animals were inoculated with 2M of Breast cells (ZR751) in theright flank. Tumor volume was monitored 3 times per week using acaliper. Treatments were initiated when a palpable mass was identified[approximately 65 mm³]. Tumor volume was compared 145 days posttreatment initiation. Animals treated with mKD/NP demonstrated asignificant lower tumor volume compared to controls (**, p<0.01,t-test).

FIG. 53: Effect of mKD/NP on breast cancer—progression free survival.NOD/SCID animals were inoculated with 2M of Breast cells (ZR751) in theright flank. Tumor volume was calculated 3 times per week and the timefrom a barely palpable tumor [approximately 65 mm³] to a tumor of asignificant size [300 mm³] was calculated. Treatments were initiated atpalpation [volume approximating 65 mm³] time. Animals treated withmKD/NP demonstrated a significant increased progression free survivaltime (time during which tumor volume is maintained lower than 300 mm³)compared to controls (**, p<0.01, t-test).

FIG. 54: Effect of mKD/NP on breast cancer—KM curve. NOD/SCID animalswere inoculated with 2M of Breast cells (ZR751) in the right flank.Tumor volume was monitored 3 times per week using a caliper. Treatmentswere initiated when a palpable mass was identified [approximately 65mm³]. Animals were sacrificed when they reached endpoint (1000 mm³). Thefraction of animals living as a function of time is represented usingKaplan-Meier survival curves. Animals treated with mKD/NP demonstrated asignificant improvement over controls (**, p<0.01, Log rank test). Thesedata demonstrate that mKD/NP is an effective treatment for breastcancer.

FIG. 55: Effect of mKD/NP on apoptosis of hGB cells (in vivo). NOD/SCIDanimals were inoculated with 1M hGB cells in the right flank. Tumorvolume was monitored 3 times per week using a caliper. Treatments wereinitiated when a palpable mass was identified [approximately 65 mm³].Animals were sacrificed when they reached endpoint (1500 mm³) and thetumors were harvested and prepared to quantify tumor cell death usingDAPI labeling to identify the SubG1 area indicative of the apoptoticfraction. The percentage of apoptotic cells was quantified using flowcytometry. Animals treated with mKD/NP demonstrated a significantincrease in cell death compared to controls (**, p<0.001 t-test). Thesedata indicate that in vivo mKD/NP increases death of hGB cells.

FIG. 56: Effect of mKD/NP on tumor initiating cells (in vivo). NOD/SCIDanimals were inoculated with 1M hGB cells in the right flank. Tumorvolume was monitored 3 times per week using a caliper. Treatments wereinitiated when a palpable mass was identified [approximately 65 mm³].Animals were sacrificed when they reached endpoint (1500 mm³) and thetumors were harvested and prepared to quantify tumor cells expressingCD133. CD133 is a marker of tumor initiating cells. The percentage ofCD133 immunoreactive cells was quantified using flow cytometry. Sub-Qtumors derived from animals treated with mKD/NP demonstrated a loweramount of CD133+ve cells compared to controls. These data indicate thatmKD/NP can target in vivo cancer stem cell (i.e. tumor initiatingcells).

FIG. 57: Effect of mKD/NP on DNA damage in CSC (in vivo). NOD/SCIDanimals were inoculated with 1M hGB cells in the right flank. Tumorvolume was monitored 3 times per week using a caliper. Treatments wereinitiated when a palpable mass was identified [approximately 65 mm³].Animals were sacrificed when they reached endpoint (1500 mm³) and thetumors were harvested and prepared to quantify tumor cells colabled withCD133 and the phosphorylated form of H2AX (pH2AX). pH2AX is a marker ofDNA double strand breaks. The percentage of CD133/pH2AX doubleimmunoreactive cells was quantified using flow cytometry. Sub-Q tumorsderived from animals treated with mKD/NP demonstrated an increase in thenumber of cancer stem cells (CD133+) exhibiting DNA damages (pH2AX+)compared to controls. These results show that mKD/NP can specificallytarget in vivo and induce DNA damages in cancer stem cells.

FIGS. 58A-58B: Effect of mKD/NP on YB1 proliferation—MGMT independentsensitization of TMZ resistance (in vivo). NOD/SCID animals wereinoculated with 1M hGB cells in the right flank. Tumor volume wasmonitored 3 times per week using a caliper. Treatments were initiatedwhen a palpable mass was identified [approximately 65 mm³]. Animals weresacrificed when they reached endpoint (1500 mm³) and the tumors wereharvested and prepared to quantify by flow cytometry the level ofexpression and phosphorylation of the Y-box binding protein 1 (YB1).YB-1 is upregulated in many human malignancies including hGB and isimplicated in the maintenance of CSCs (Fotovati et al., 2011). Not onlyis YB-1 critical for hGB maintenance and proliferation but it also playsa role in the mechanism (MGMT independent) of resistance to TMZ byrepairing the DNA damages caused by the chemotherapy drug (Gao et al.,2009). Hence, targeting YB-1 represents an appealing approach to inhibithGB proliferation and to sensitize hGB to TMZ. Compared to controls,tumor derived from animals treated with mKD/NP demonstrated asignificant decrease of YB1 level of expression (A) as well as ofphosphorylation (B). * p<0.05, F-test. These results demonstrate thatmKD/NP can target the YB-1 pathway, proposing a mechanism by which thetreatment is inhibiting tumor cell proliferation and is sensitizing thecells to TMZ via a mechanism independent to MGMT.

FIG. 59: Effect of mKD/NP on inhibiting chemoresistance in CSC (invivo). NOD/SCID animals were inoculated with 1M hGB cells in the rightflank. Tumor volume was monitored 3 times per week using a caliper.Treatments were initiated when a palpable mass was identified[approximately 65 mm³]. Animals were sacrificed when they reachedendpoint (1500 mm³) and the tumors were harvested and prepared toquantify tumor cells colabled with CD133 and NFkB. NFkB is ananti-apoptotic effector that also contribute to chemoresistance in hGB(Han et. al., 2004). A) The percentage of CD133/NFkB doubleimmunoreactive cells was quantified using flow cytometry. Tumor derivedfrom animals treated with mKD/NP demonstrated a decrease in the numberof cancer stem cells (CD133+) positive for NFkB compared to controls.These results suggest that mKD/NP can target CSCs by inhibiting theanti-apoptotic effector NFkB.

FIG. 60: Effect of mKD/NP on MGMT expression (in vitro). Patient derivedGB cells were treated in vitro with TMZ [10 μM] or TMZ [10 μM]& NP (EGCG(8 μM), Curcumin (0.5 μM) and sulforaphane (2.5 μM)). After seven daysof treatment cells were harvested and analyzed for expression level ofMGMT using flow cytometry. The median fluorescence in the two treatedgroups were compared relative to the untreated controls. The dataindicates that TMZ induces nearly a 30% increase in MGMT expression andthat NP is able to attenuate this increase to near control levels.

FIGS. 61A-61B: Effect of mKD/NP on surviving expression (in vitro). A)hGB cells were cultured in the neurosphere assay. After 7 days in vitro,the cells were processed for immunolabeling and flow cytometry analysisto access Survivin (a member of the inhibitor of apoptosis for which theoverexpression is associated with chemoresistance) level. Expressionlevel of Survivin is increased with TMZ treatment (10 μM, dailytreatment for 7 days). This effect is reduced to control levels when TMZtreated cells are also exposed to NP [EGCG (8 μM), Curcumin (0.5 μM) andsulforaphane (2.5 μM), daily treatment for 4-7 days]. B) Similarly, NPreduces the fraction of cells expressing Survivin that is increased whencell are treated with TMZ. **, ***, compared to control; ^(###),compared to TMZ. 1-way ANOVA, 2 symbols p<0.01, 3 symbols p<0.0001.

FIGS. 62A-62B: mKD/NP sensitization of tumor cells to TMZ via MGMTdependent mechanism (in vivo). NOD/SCID animals were inoculated with 1MhGB cells in the right flank. Tumor volume was monitored 3 times perweek using a caliper. Treatments were initiated when a palpable mass wasidentified [approximately 65 mm³]. Animals were sacrificed when theyreached endpoint (1500 mm³) and the tumors were harvested and preparedto quantify tumor cells expressing MGMT and pSTAT3. A) MGMT providesresistance to TMZ. The percentage of MGMT immunoreactive cells wasquantified using flow cytometry. Animals treated with mKD/NPdemonstrated a decrease of MGMT+ve cells compared to controls. p<0.05,t-test. B) Phosphorylation of Stat3 has been correlated with TMZresistance through a mechanism dependent to MGMT (Kohsaka et al., 2012).The ability of mKD/NP to decrease Stat3 activation and MGMT expressionprovide a potential mechanism by which mKD/NP sensitize tumor cells toTMZ (described in FIG. 29) via a MGMT dependent mechanism.

FIG. 63: Effect of mKD/NP on Zeb1 expression (in vivo). NOD/SCID animalswere inoculated with 1M hGB cells in the right flank. Tumor volume wasmonitored 3 times per week using a caliper. Treatments were initiatedwhen a palpable mass was identified [approximately 65 mm³]. Animals weresacrificed when they reached endpoint (1500 mm³) and the tumors wereharvested and prepared to quantify tumor cells expressing ZEB1. ZEB1, amarker of tumor initiating cells, is an important candidate molecule forhGB recurrence, a marker of invasive tumor cells and a potentialtherapeutic target (Siebzehnrubl et al., under review). The percentageof ZEB1 immunoreactive cells was quantified using flow cytometry. Tumorsderived from animals treated with mKD/NP demonstrated a significantdecrease of ZEB1+ve cells compared to controls (*, p<0.05, t-test).

FIG. 64: Effect of mKD/NP on NFkB expression (in vivo). NOD/SCID animalswere inoculated with 1M hGB cells in the right flank. Tumor volume wasmonitored 3 times per week using a caliper. Treatments were initiatedwhen a palpable mass was identified [approximately 65 mm³]. Animals weresacrificed when they reached endpoint (1500 mm³) and the tumors wereharvested and prepared to quantify by flow cytometry the percentage oftumor cells expressing NFkB. NFkB is an anti-apoptotic effector thatalso contribute to chemoresistance in hGB (Han et. al., 2004). Tumorsderived from animals treated with mKD/NP demonstrated a significantdecrease of NFkB+ve cells compared to controls (*, p<0.05, t-test).

FIG. 65: Effect of mKD/NP on Glut3 expression (in vivo). Many tumorcells display increased level of glucose metabolism compared with normalcells. Glucose uptake is mediated by the glucose transporter (GLUT)family including Glut 3 that has been reported to be upregulated inglioblastoma (Boado et al., 1994) and to participate in theproliferation of tumor cells as well as in the acquired resistance toTMZ (Le Calve et al., 2010). These findings suggest that selectivetargeting of Glut3 would delay tumor cells proliferation and developmentof TMZ resistance. NOD/SCID animals were inoculated with 1M hGB cells inthe right flank. Tumor volume was monitored 3 times per week using acaliper. Treatments were initiated when a palpable mass was identified[approximately 65 mm³]. Animals were sacrificed when they reachedendpoint (1500 mm³) and the tumors were harvested and prepared toquantify by flow cytometry the percentage of tumor cells expressingGlut3. Tumors derived from animals treated with mKD/NP demonstrated asignificant decrease of Glut3+ve cells compared to controls (**,p<0.005, t-test).

FIGS. 66A-66D: mTor pathway—In Vivo. mTor pathway is activated by growthfactors, nutrients, energy and stress signals and is implicated in thecontrol of cell growth, proliferation and survival. Deregulation of mTorpathway (e.g. up regulation of upstream activator such as AKT ordownstream effectors such as S6 and 4EBP) has been reported in manycancers. mTor pathway is activated by the PI3K/AKT pathway. mToractivation leads to phosphorylation/activation of S6 andphosphorylation/inactivation of 4EBP, the 2 best characterizeddownstream effectors of mTor regulating ribosomal biogenesis andproteins synthesis respectively. Therefore mTor is an appealingtherapeutic target and mTor pathway inhibitors represent potentialcandidates for viable therapeutic strategies. A) NOD/SCID animals wereinoculated with 1M hGB cells in the right flank. Animals were sacrificedwhen they reached endpoint (1500 mm3) and the tumors were harvested andprepared to quantify by flow cytometry the percentage of tumor cellsexpressing p-S6. Animals treated with mKD/NP demonstrated a significantdecrease of pS6+ve cells compared to controls. (**, p<0.005, t-test). B)Tissue specimens were harvested at endpoint and pS6 immunolabeling wascompared between controls and mK/NP treated animals. Micrographsdemonstrated a decreased pS6 staining in tumor treated with mKD/NP. C)NOD/SCID animals were inoculated with 1M hGB cells in the right flank.Animals were sacrificed when they reached endpoint (1500 mm3) and thetumors were harvested and prepared to quantify by flow cytometry thepercentage of tumor cells expressing p4ABP1. Animals treated with mKD/NPdemonstrated a significant decrease of p4ABP1+ve cells compared tocontrols. (**, p<0.005, t-test). D) WB: pS6 & p4EBP1. Decreasedexpression of pS6 and p4ABP1 in the mKD/NP treated tumor wasdemonstrated by also Western Blot.

DETAILED DESCRIPTION OF THE INVENTION

A typical North American diet provides approximately 50 to 60% of itscaloric intake from carbohydrates. As carbohydrates are the main sourceof glucose and the primary source of energy for glucose stored tumorcells, reducing carbohydrates through dietary restrictions can assist inlowering glucose levels and hence limiting tumor cell access to thisfuel source. Thus, one aspect of the disclosed invention pertains tomethods of treating a proliferative disorder in a subject comprising theadministration, to the subject, of a composition comprising one or morecomponent(s) selected from: epigallocatechin-3-gallate (EGCG); curcumin;glucosinolates and/or derivatives thereof, such as glucoraphanin and/orsulforaphane (SFN) (optionally in the form of broccoli sprouts, thesprouts of other cruciferous vegetables or cruciferous vegetablesthemselves) and, optionally, Daikon radish (in the form of matureradish, sprouts or powders of the radish, spouts or extracts therof)alone or in combination with a low carbohydrate diet. The components canbe administered individually as separate compositions or in variouscombinations (e.g., pairs, three-component compositions or a singlecomposition containg all four or five components).

MCTs are fractionated from coconuts or palm kernel oils and are usedclinically for patients with malabsorption symptoms. Due to their smallmolecular size MCT are digested rapidly traveling directly to the liverwhere they are quickly metabolized and lead to elevated blood ketoneslevels. Increased ketone and reduced glucose concentrations are theprimary physiological effects of a ketogenic diet (for example, a dietcomposed of 90% fat and 10% proteins/carbohydrates).

EGCG is the most abundant catechin found in green tea. Curcumin isderived from turmeric.

Cruciferous vegetables contain a group of substances known asglucosinolates, which are sulfur-containing chemicals. During digestion,food preparation or chewing the glucosinolates are broken down into anumber of biologically active compounds, these include, but are notlimited to, indoles, nitriles, thiocyanates, isothiocyanates,Indole-3-carbinol and sulforaphane [SFN].

SFN is a bioactive molecule derived from the conversion of aglucosinolate precursor, glucoraphanin, found in cruciferous vegetables(for example, Brussels sprouts, cabbage, cauliflower, bok choy, kale,collards, Chinese broccoli, broccoli raab, kohlrabi, mustard, turnip,radish, arugula, and watercress). It is found in highest concentrationin broccoli sprouts. Effective doses of glucosinolates, such asglucoraphanin and its biologically active breakdown products includingSFN can be delivered by consumption of sprouts or sprout powders derivedfrom the aforementioned cruciferous vegetables or plants from the genusBrassica. The phrases “composition(s) comprising glucosinolates and/orderivatives thereof, such as glucoraphanin and/or sulforaphane (SFN)” or“composition(s) comprising glucosinolates” or “composition(s) comprisingglucoraphanin” or “composition(s) comprising SFN” may comprise one ormore powders of mature plants of the genus Brassica or maturecruciferous vegetables, consumable vegetative matter of mature plants ofthe genus Brassica or mature cruciferous vegetables, dehydrated ornon-dehydrated sprouts of plants of the genus Brassica or sprouts ofcruciferous vegetables, or powdered sprouts obtained from cruciferousvegetables or from plants of the genus Brassica.

In some embodiments, the composition(s) comprising glucosinolates and/orderivatives thereof, such as glucoraphanin and/or sulforaphane (SFN)comprise powders of mature plants of the genus Brassica or maturecruciferous vegetables, consumable vegetative matter of mature plants ofthe genus Brassica or mature cruciferous vegetables, powders formed fromdehydrated or non-dehydrated sprouts of plants of the genus Brassica orsprouts of cruciferous vegetables, or powdered sprouts obtained fromcruciferous vegetables or from plants of the genus Brassica. Asdiscussed above, powders from one or more cruciferous vegetable orplants from the genus Brassica can be combined into a compositioncomprising glucosinolates and/or derivatives thereof, such asglucoraphanin and/or sulforaphane (SFN). The powders discussed above maybe provided in the form of freeze-dried powders. The administration ofsuch powders delivers glucosinolates, including glucoraphanin, acompound subsequently metabolized to SFN by myrosinase, to the subjectbeing treated. Daikon radish can be combined in with the componentsdiscussed above into compositions. Where Daikon radish is formulatedinto a composition comprising the various components discussed herein,it can be provided in the form of a powder (optionally freeze-dried),sprout, mature vegetable or a sprout powder (including dehydrated and/orfreeze-dried sprout powders).

The current invention is directed to a treatment of proliferativedisorders, for example, cancer. Thus, one aspect of the currentinvention provides a treatment of a proliferative disorder comprisingadministering to a subject in need of a treatment for the proliferativedisease, a combination of compounds and, optionally, simultaneouslyproviding the subject with a low carbohydrate diet or a modifiedketogenic diet or a ketogenic diet. The combination of compounds to beadministered to the subject comprise EGCG, curcumin, compositionscomprising glucosinolates such as glucoraphanin and breakdown productssuch as SFN (these can be derived from broccoli sprouts or sprouts ofother cruciferous vegetables or plants of the genus Brassica), and,optionally, Daikon radish sprout, a Daikon radish sprout extract or apowder of said extract, the Daikon radish or the Daikon radish sprout.These compounds (components) can be administered as a single compositionor individually (as separate compositions/components) sequentially orsimultaneously. This aspect of the invention can also provide for therestoration of normal cell proliferation for the proliferative disorderbeing treated. Thus, for various forms of cancer treated in this aspectof the invention, excessive cell proliferation associated with theproliferative disorder can be reduced or attenuated to levels at, ornear normal proliferative levels for the particular cell giving rise tothe proliferative disorder (e.g. a B-cell if B-cell lymphomas are beingtreated). Normal or near normal proliferative levels of cells are,generally, known in the art or can be dertermined by those skilled inthe art. Thus, certain embodiments of the invention provide forreducing/attenuating the proliferation cells associated with aproliferative disorder by at least about 50%, 60%, 70%, 80%, 95% ormore.

Various other aspects of the invention provide for the use of acomposition comprising epigallocatechin-3-gallate, a compositioncomprising curcumin, and a composition comprising glucosinolates such asglucoraphanin and breakdown products such as SFN, and, optionally, amodified ketogenic diet or a ketogenic diet in subjects for thetreatment of a proliferative disorder, decreasing the incidence of aproliferative disease in a subject, slowing the progression of aproliferative disease in a subject, increasing survival in a subjecthaving a proliferative disease, enhancing the effect of conventionaltherapies for patients with proliferative diseases, sensitizingresistant cells to conventional therapies for patients withproliferative disease, reducing neuronal effects of chemotherapy in asubject treated with a chemotherapeutic regimen or reducingdownregulation of neural stem cells (NSC) of the CNS in a subjectdeveloping a tumor or having a tumor or in a subject having aneurodegenerative disease or disorder. The compositions described abovecan be administered separately, separately in two, three or fourcomponent compositions or as a combined composition of all thecomponents of the composition (including, optionally, Daikon radishsprouts, extracts thereof and/or powders thereof as described above).Slowing the progression of a proliferative disease in a subject relatesto reducing the speed of a proliferative disease to advance overtime andcan be measured, for example, as a reduction in increased volume of atumor. Increasing survival in a subject having a proliferative diseaserelates to delaying the progression of proliferative diseases, in asubject and leads to an increased in the time of survival of a subjecthaving a proliferative disease. Enhancing the effect of conventionaltherapies for patients with proliferative diseases relates to combiningthe disclosed method with conventional therapies used to treat subjectswith proliferative diseases. This combination of therapies leads topositive outcomes equal to (additive effect) or greater than(synergistic effect) the individual effect of the conventional treatmentand the disclosed method. Sensitizing resistant cells to conventionaltherapies for patients with proliferative disease relates to the abilityof the disclosed method to convert proliferative diseases that wereinsensitive to conventional treatments used to treat patients withproliferative disease into proliferative diseases that respond to theconventional treatment to which the disease was, previously,unresponsive/refractory. Reducing neuronal effects of chemotherapyrelates to reducing or preventing the decline of neural stem andprogenitor cell activity observed when a subject undergoes chemotherapy.

The modified ketogenic diet is a diet that contains at least 5% and nomore than about 20% carbohydrates (as a function of total caloric intakeby the intake by the subject each day) and the balance of the diet forthe subject comprises fats and proteins. Thus, the diet can, as afunction of total caloric intake each day, contain about 5% to about 20%carbohydrates, about 30% to about 75% fats and about 5% to about 65%proteins. In certain embodiments, the diet can provides between about 8%and about 15% carbohydrates, about 50% to about 70% fats and about 18%to about 42% proteins. In some embodiments, from about 30% to about 70%(e.g., about 30%, about 40%, about 50%, about 60% or about 70%) of thefat content of the subject's diet can be made up of medium chaintriglycerides (MCT). Other embodiments provide that MCT make up about50% of the fat content of the subject's diet.

As a function of total amount of food (grams) based on a daily intake of2000 kilocalories (and based on the fact that 1 g of carbohydratesprovides 4 kilocalories, 1 g of fat provides 9 kilocalories, 1 g ofproteins provides 4 kilocalories and 1 g of MCTs provide 6.8kilocalories) the modified ketogenic diet is a diet that contains atleast 25 g and no more than 100 g of carbohydrates and the balance ofthe diet for the subject comprises fats and proteins. Thus, the dietcan, as a function of total grams intake each day, contain about 25 g to100 g of carbohydrates, about 67 g to about 167 g of fats and about 25 gto about 325 g of proteins. In certain embodiments, the diet canprovides between about 40 g and about 75 g of cabohydrates, about 111 gto about 155 g of fats and about 90 g to about 210 g of proteins. Insome embodiments, from about 30% to about 70% (e.g., about 30%, about40%, about 50%, about 60% or about 70%) of the fat content of thesubject's diet can be made up of medium chain triglycerides (MCT). Thisrepresents from about 40 g to about 165 g of MCTs.

The ketogenic diet (KD) is a diet wherein the carbohydrate content isless than, or equal to, about 5% of the total caloric intake the subjecteach day and the balance of the diet consists of fats or proteins. Thus,the diet provides, as a function of total caloric intake each day, about5% or less carbohydrate, about 30% to about 90% fat and about 5% toabout 70% protein. In certain embodiments, the diet provides about 3%(or less) carbohydrate, about 57% to about 95% fat, about 5% to about40% protein. In some embodiments, from about 30% to about 70% (e.g.,about 30%, about 40%, about 50%, about 60% or about 70%) of the fatcontent of the subject's diet can be made up of medium chaintriglycerides (MCT). Other embodiments provide that MCT make up about50% of the fat content of the subject's diet.

In another embodiment of the invention, the treatment comprisesproviding, to a subject in need of a treatment for a proliferativedisorder, a mKD or KD diet and, optionally, administering a compositioncomprising one or more of EGCG, curcumin, glucosinolates and,optionally, Daikon radish sprout. Various embodiments provide for theadministration of a composition comprising EGCG, curcumin andglucosinolates or a composition comprising EGCG, curcumin,glucosinolates and Daikon radish sprout to the subject. In any aspect ofthe invention, the composition comprising one or more of EGCG, curcumin,compositions comprising glucosinolates such as glucoraphanin and and itsbreakdown product SFN (which are found in high concentrations inbroccoli sprouts or sprouts of other cruciferous vegetables or plants ofthe genus Brassica), and Daikon radish sprout or extracts thereof may beprovided in the form of a powder or an extract produced from foodproducts where at least one of these compounds are present naturally.Furthermore, compositions administered to a subject can be administeredas a combination (e.g., each of EGCG, curcumin, compositions comprisingglucosinolates such as glucoraphanin and and its breakdown product SFN,and/or Daikon radish sprout in a single composition or each of thecomponents (EGCG, curcumin, compositions comprising glucosinolates suchas glucoraphanin and and its breakdown product SFN, and Daikon radishsprout) can be provided (e.g., in the form of capsules, caplets tablets,powders, gels or other unit dosage forms) to the subject individuallyfor simultaneous or sequential consumption.

Any of the aforementioned aspects of the invention for the treatment ofa proliferative disorder or cancer may further comprise theadministration of one or more additional anticancer therapy ortherapies. Such therapies include, but are not limited to, radiotherapy,chemotherapy, surgery, immunotherapy, small molecule, kinase inhibitionand/or monoclonal antibody therapy (e.g., rituximab for the treatment ofB-cell lymphomas). In an embodiment of the invention the additionaltherapy comprises administration of temozolomide (TMZ).

In various other aspects of the invention, anti-cancer therapy ortherapies are administered in addition to (in combination with) thetreatment provided by the current invention. Such anti-cancer therapiesinclude, but are not limited to, administering one or more of:Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (PaclitaxelAlbumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC,AC-T, Adcetris (Brentuximab Vedotin), ADE, Adriamycin (DoxorubicinHydrochloride), Adrucil (Fluorouracil), Afinitor (Everolimus), Aldara(Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium),Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil),Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant,Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine),Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwiniachrysanthemi, Avastin (Bevacizumab), Axitinib, Azacitidine, BEACOPP,Bendamustine Hydrochloride, BEP, Bevacizumab, Bexarotene, Bexxar(Tositumomab and I 131 Iodine Tositumomab), Bleomycin, Bortezomib,Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Cabazitaxel,Cabozantinib-S-Malate, CAF, Campath (Alemtuzumab), Camptosar(Irinotecan, ydrochloride), Capecitabine, CAPOX, Carboplatin,CARBOPLATIN-TAXOL, Carfilzomib, CeeNU (Lomustine), Cerubidine(Daunorubicin Hydrochloride), Cervarix (Recombinant HPV BivalentVaccine), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP,Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex(Clofarabine), Clolar (Clofarabine), CMF, Cometriq(Cabozantinib-S-Malate), COPP, Cosmegen (Dactinomycin), Crizotinib, CVP(COP), Cyclophosphamide, Cyfos (Ifosfamide), Cytarabine, Cytarabine,Liposomal, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide),Dacarbazine, Dacogen, (Decitabine), Dactinomycin, Dasatinib,Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin, iftitox,Denosumab, DepoCyt (Liposomal Cytarabine), DepoFoam (LiposomalCytarabine), Dexrazoxane hydrochloride, Docetaxel, Doxil (DoxorubicinHydrochloride Liposome), Doxorubicin Hydrochloride, DoxorubicinHydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome),DTIC-Dome (Dacarbazine), Efudex (Fluorouracil), Elitek (Rasburicase),Ellence (Epirubicin Hydrochloride), Eloxatin (Oxaliplatin), EltrombopagOlamine, Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride,EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib),Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi),Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet(Doxorubicin Hydrochloride Liposome), Everolimus, Evista (RaloxifeneHydrochloride), Exemestane, Fareston (Toremifene), Faslodex(Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (FludarabinePhosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil),Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI,FOLFIRI-BEVACIZUMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV,Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gefitinib,Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, Gemtuzumab Ozogamicin,Gemzar (Gemcitabine, ydrochloride), Gleevec (Imatinib Mesylate),Glucarpidase, Halaven (Eribulin Mesylate), Herceptin (Trastuzumab), HPVBivalent Vaccine, Recombinant, HPV Quadrivalent Vaccine (Recombinant),Hycamtin (Topotecan Hydrochloride), Ibritumomab Tiuxetan, ICE, Iclusig(Ponatinib Hydrochloride), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum(Ifosfamide), Imatinib Mesylate, Imiquimod, Inlyta (Axitinib),Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax(Romidepsin), Ixabepilone, Ixempra (Ixabepilone), Jakafi (RuxolitinibPhosphate), Jevtana (Cabazitaxel), Keoxifene (Raloxifene Hydrochloride),Kepivance (Palifermin), Kyprolis (Carfilzomib), Lapatinib Ditosylate,Lenalidomide, Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil),Leuprolide Acetate, Levulan (Aminolevulinic (Acid), Linfolizin(Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), LiposomalCytarabine, Lomustine, Lupron (Leuprolide Acetate), Lupron Depot(Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), LupronDepot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (LeuprolideAcetate), Marqibo (Vincristine Sulfate Liposome), Matulane (ProcarbazineHydrochloride), Mechlorethamine Hydrochloride, Mesna, Mesnex (Mesna),Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF(Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate),Mitomycin C, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor),Mustargen (Mechlorethamine hydrochloride), Mutamycin (Mitomycin C),Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), NanoparticlePaclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation),Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide),Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilotinib, Nolvadex(Tamoxifen Citrate), Nplate (Romiplostim), Ofatumumab, Omacetaxine,Mepesuccinate, Oncaspar (Pegaspargase), Ontak (Denileukin Diftitox),Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized NanoparticleFormulation, Palifermin, Palonosetron Hydrochloride, Panitumumab,Paraplat (Carboplatin), Paraplatin (Carboplatin), PazopanibHydrochloride, Pegaspargase, Pemetrexed Disodium, Perjeta (Pertuzumab),Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor,Ponatinib Hydrochloride, Pralatrexate, Prednisone, ProcarbazineHydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta(Eltrombopag Olamine), Provenge (Sipuleucel-T), Raloxifenehydrochloride, Rasburicase, R-CHOP, R-CVP, Recombinant HPV BivalentVaccine, Recombinant HPV, Quadrivalent Vaccine, Regorafenib, Revlimid(Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab),Rituximab, Romidepsin, Romiplostim, Rubidomycin (DaunorubicinHydrochloride), Ruxolitinib Phosphate, Sclerosol Intrapleural Aerosol(Talc), Sipuleucel-T, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORDV, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib),Sunitinib Malate, Sutent (Sunitinib Malate), Synovir (Thalidomide),Synribo (Omacetaxine Mepesuccinate), Talc, Tamoxifen Citrate, TarabinePFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin(Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere(Docetaxel), Temodar (Temozolomide), Temozolomide, Temsirolimus,Thalidomide, Thalomid (Thalidomide), Toposar (Etoposide), TopotecanHydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and I 131Iodine Tositumomab, Totect (Dexrazoxane Hydrochloride), Trastuzumab,Treanda (Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide),Tykerb (Lapatinib Ditosylate), Vandetanib, VAMP, Vectibix (Panitumumab),VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar(Vinblastine Sulfate), Vemurafenib, VePesid (Etoposide), Viadur(Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate,Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, VincristineSulfate Liposome, Vinorelbine Tartrate, Vismodegib, Voraxaze(Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride),Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda(Capecitabine), XELOX, Xgeva (Denosumab), Xtandi (Enzalutamide), Yervoy(Ipilimumab), Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib), Zevalin(Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride),Ziv-Aflibercept, Zoledronic Acid, Zolinza (Vorinostat), Zometa(Zoledronic Acid), and Zytiga (Abiraterone Acetate).

In certain aspects of the invention the proliferative disease to betreated by the current invention is not glioblastoma. However, thetreatment of the current invention provides a meaningful treatment for awide variety of other proliferative disorders. For example, thetreatment provided by the current invention appears to be effective andnon-toxic in preclinical models, which included brain, breast, colon,and lung cancer. Thus, the proliferative disorders that can be treatedwith the treatment of current invention include, but are not limited to,Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, AdrenocorticalCarcinoma, AIDS-Related Cancers, AIDS-Related Lymphoma, Anal Cancer,Appendix Cancer, Astrocytoma, Cerebellar Astrocytoma, Basal CellCarcinoma, Bile Duct Cancer, Extrahepatic Bladder Cancer, BladderCancer, Bone Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma,Embryonal Tumors, Cerebral Astrocytoma, Ependymoblastoma,Medulloblastoma, Medulloepithelioma, Pineal Parenchymal Tumors ofIntermediate Differentiation, Supratentorial Primitive NeuroectodermalTumors and Pineoblastoma, Visual Pathway and Hypothalamic cancer, Brainand Spinal Cord Tumors, Breast Cancer, Bronchial Tumors, BurkittLymphoma, Carcinoid Tumor, Gastrointestinal Cancer, Carcinoma of Headand Neck, Central Nervous System Lymphoma, Cervical Cancer, ChronicLymphocytic Leukemia, Chronic Myelogenous Leukemia, ChronicMyeloproliferative Disorders, Colorectal Cancer, Cutaneous T-CellLymphoma, Endometrial Cancer, Ependymoblastoma, Ependymoma, EsophagealCancer, Ewing Family of Tumors, Extracranial Germ Cell Tumor,Extrahepatic Bile Duct Cancer, Eye Cancer, Intraocular Melanoma,Retinoblastoma, Gallbladder Cancer, Gastric (Stomach) Cancer,Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor (GIST),Extracranial Germ Cell Tumor, Germ Cell Tumor, Extragonadal Germ CellTumor, Ovarian Cancer, Gestational Trophoblastic Tumor, Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer,Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Hypopharyngeal Cancer,Intraocular Melanoma Islet Cell Tumors (Endocrine Pancreas), KaposiSarcoma, Kidney (Renal Cell) Cancer, Kidney Cancer, Laryngeal Cancer,Chronic Lymphocytic Leukemia, Chronic Leukemia, Myelogenous Leukemia,Lip and Oral Cavity Cancer, Lung Cancer, Non-Small Cell Lung Cancer,Small Cell Lymphoma, Cutaneous T-Cell Lymphoma, Non-Hodgkin Lymphoma,Macroglobulinemia, Waldenström, Malignant Fibrous Histiocytoma of Boneand Osteosarcoma, Medulloblastoma, Medulloepithelioma, Melanoma,Intraocular Merkel Cell Carcinoma, Mesothelioma, Metastatic SquamousNeck Cancer with Occult Primary, Mouth Cancer, Multiple EndocrineNeoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm, MycosisFungoides, Myelodysplastic Syndromes, Myelodysplastic/MyeloproliferativeDiseases, Myelogenous Leukemia, Multiple, Myeloproliferative Disorders,Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal CancerNeuroblastoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral CavityCancer, Lip and Oropharyngeal Cancer, Osteosarcoma and Malignant FibrousHistiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ CellTumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer,Pancreatic Cancer, Islet Cell Tumors, Papillomatosis, Paranasal Sinusand Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, PharyngealCancer, Pheochromocytoma, Pineal Parenchymal Tumors of IntermediateDifferentiation, Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Pleuropulmonary Blastoma, Primary Central Nervous SystemLymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer,Renal Pelvis and Ureter Caner, Transitional Cell Cancer, RespiratoryTract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma,Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Ewing Family of TumorsSarcoma, Kaposi Sarcoma, Soft Tissue Sarcoma, Uterine Sézary Syndrome,Skin Cancer (Nonmelanoma), Skin Carcinoma, Merkel Cell, Small Cell LungCancer, Small Intestine Cancer, Squamous Cell Carcinoma, Squamous NeckCancer with Occult Primary Cancer, Supratentorial PrimitiveNeuroectodermal Tumors, T-Cell Lymphoma, Mycosis Fungoides and SézarySyndrome, Testicular Cancer, Throat Cancer, Thymoma and ThymicCarcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvisand Ureter, Gestational Trophoblastic Tumor, Carcinoma of UnknownPrimary Site, Urethral Cancer, Uterine Cancer, Endometrial UterineSarcoma, Vaginal Cancer, Vulvar Cancer, Waldenström Macroglobulinemia,and Wilms Tumor.

A further aspect of the invention provides a composition comprising MCT,ECGC, curcumin, and compositions comprising glucosinolates and/orderivatives thereof, such as glucoraphanin and/or sulforaphane (SFN) inthe form of a powder, drink, emulsion, gel, or mixture thereof. An evenfurther aspect of the invention provides a composition comprising MCT,ECGC, curcumin, compositions comprising glucosinolates and/orderivatives thereof, such as glucoraphanin and/or sulforaphane (SFN),and Daikon radish sprout or extracts of ECGC, curcumin, compositionscomprising glucosinolates and/or derivatives thereof, such asglucoraphanin and/or sulforaphane (SFN), and Daikon radish thereof inthe form of a capsule, tablet, powder, drink, emulsion, gel, or mixturethereof. A subject in need of a treatment for a proliferative disease orsparing of NSC downregulation may ingest the composition provided by thecurrent invention either directly or by mixing it with other foods ordrinks, for example, water, fruit juice, yogurt, soups, stews, pasta,etc. Further, the composition (or individual components) can also beincorporated into other food products, for example, cake, cookies,cereal bars, etc.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all Figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples, which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight or by calories and all solvent mixtureproportions are by volume unless otherwise noted.

EXAMPLE 1 The Modified Ketogenic Diet [mKD] Alters Glucose and KetoneLevels to the Same Extent as the Ketogenic Diet [KD]

NOD-SCID animals were placed on a KD or mKD or mKD+EDP for two weeks.The compositions of the diets are as follows (expressed as percentage ofcalories):

Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

KD: 92% Fat, 3% carbohydrate, 5% protein.

mKD: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein.

NP diet: 55% carbohydrate, 30% protein, 15% fat+Sulforaphane (SFN; 25mg/kg; BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Ten animals, in each dietary group, were placed on the modified diet for2 weeks at which point tail tip method was used to collect blood via thefollowing protocol: “Using a 50 mL conical (or mouse restrainer) place anon-anesthetized mouse gently inside grasping the mouse by the tail.Place the mouse's tail on a hard surface. Using a scalpel cut off lessthan 1 mm of the tip of the tail. Place your fingers at the base of thetail and gently squeeze upward running your fingers from base to tip ofthe tail. One to two drops of blood (5-10 μL) will appear at the tip ofthe tail. Using a dry gauze wipe away first few drops of blood andrepeat steps until desired amount of blood is collected (should be dark,whole blood, clear [plasma like] blood will give you an inconsistentblood reading). Place the mouse back into the cage and monitor forexcess bleeding.”

Blood samples were analyzed with Precision Xtra blood glucose/ketonemonitor and expressed in mg/dl and mmol for glucose and ketones,respectively. With regards to glucose levels, all 4 experimental dietsresulted in a significant reduction in glucose [FIG. 1, p<0.001].Similarly, ketone levels were elevated in all the dietary groups exceptfor the NP diet [FIG. 2, p<0.001]. These data demonstrate that the mKDand mKD/NP diet is able to mimic the two key physiological features ofthe KD, a significant reduction in glucose and a significant increase inketones.

EXAMPLE 2 The mKD/NP Diet is Safe and has No Signs of Toxicity

The vast majority of cancer therapeutics has dose-limiting toxic sideeffects that impact not only a patient's well-being but it also resultsin suspended treatments and reduced dosages that can impact treatmentefficacy. One of the best indicators of overall health in a rodent isits body weight [this is true in human patients as well]. NOD-SCIDanimals received 1M GB cells [patient derived lines] into the rightflank. Animals were monitored 3 times per week for signs of tumorformation. Once a tumor was identified [by palpation & approximately 65mm3]] animals were randomly assigned to 1 or 2 groups: [1] Control dietor [2] mKD/NP diet. Body weight was monitored 3 times per week. FIG. 3depicts percentage change in body weight till the first control animalreached endpoint [16 days]. While the mKD/NP group initially lost weightover the first few days [due to adjusting to a new diet], they quicklyrecovered the lost weight and continued to gain weight at asignificantly greater rate than the control group [p<0.005, linearregression, GraphPad].

When animals reached endpoint blood was collected via intracardiac orretro-orbital puncture and blood samples were sent to ComprehensiveClinical Pathology Services, LLC for the following analysis:

[1] Alkaline phosphatase [ALP]—liver, bones and pancreas function test

[2] Alanine transaminase [ALT]—liver function test

[3] Aspartate animotransferase [AST]—liver function test

[3] Creatinine—kidney function test

FIG. 4 reveals no statistically significant differences in liver,kidney, bones and pancreas function between the control and mKD/NP feedanimals. Together, FIGS. 3 and 4, support the conclusion that mKD/NPdiet is safe and has no noted toxic side effects.

EXAMPLE 3 Comparison of Safety Between Standard of Care [SOC] vs. mKD/NP

The first line chemotherapy for high grade gliomas is temozolomide[TMZ], which is an oral alkylating agent that damages DNA and triggerscell death. While TMZ is an effective chemotherapy agent it also hassignificant toxicity. Using the same subcutaneous model as noted inexample 2, at the time of tumor presentation animals were randomlyassigned to one of three groups: [1] control diet, no treatment; [2]control diet, SOC [TMZ, 20 mg/kg three times/week]; [3] mKD/NP diet. Weanalyzed the number of animals that died as a result of non-tumorrelated causes and noted that SOC treatment resulted in an increase inmortality compared to the untreated controls [FIG. 5]. A similarincrease in mortality was not seen in the mKD/NP group, where the deathrate was not statistically different than the controls but wassignificantly lower than the SOC group.

While receiving SOC treatment, body weight was measured and notsurprisingly was significantly reduced in the SOC group relative to thecontrol and mKD/NP treated animals [FIG. 6]. It is important to notethat mKD/NP treatment was as effective as SOC [see example 11]. Hence,these data teach that mKD/NP has no adverse effects on overall health[as defined by body weight] and does not directly affect mortality,unlike SOC that affects overall health and results in increasedmortality.

EXAMPLE 4 Natural Products [NP] Provide Effective Cancer Treatment

Patient derived hGB cells were cultured under defined conditions usingestablished protocols that allow the tumor cells to retain both theirphenotypic and genotypic properties in vitro. hGB cell lines weretreated with the following NPs:

[1] EGCG—8 μM

[2] Curcumin—0.5 μM

[3] SFN—2.5 μM

[4] a combination of all three NPs

Each of the NPs resulted in a significant reduction in the overallnumber of cells that were produced relative to the control cultures[FIG. 7]. However, the simultaneous application of all three NPsresulted in a synergistic effect with regards to reducing the productionof new cells. These data support the hypothesis that each of the NPsexert their effect by different mechanisms.

One million hGB cells were implanted into the right flank of NOD-SCIDmice, and at the time that a palpable tumor was identified the animalswere randomized into one of 5 groups:

[1] Control diet

[2] Control diet+EGCG [1200 mg/kg]

[3] Control diet+Curcumin [1200 mg/kg]

[4] Control diet+SFN [25 mg/kg; BSP95%/DRSP5%]

[5] Control diet+EGCG/Curcumin/SFN [same concentrations as group 2-4]

Animals were kept on their respective diets till tumors reached endpoint[1500 mm3] and they were killed. Kaplan Meier survival curves weregenerated using GraphPad. While groups 2-4 did not live longer than thecontrol animals, those treated with all three natural products [Group 5]did live significantly longer [FIG. 8]. These data support thesynergistic actions of combining the three NPs and their effect onreducing tumor progression and enhancing lifespan.

Together the data teaches that our unique combination of NPs has ansynergistic effect in vitro but a synergistic action in vivo, indicatingthat this particular combination produces an unexpected anti-cancereffect and as such is defined as a unique polymolecular botanical drug.

EXAMPLE 5 Natural Products [NPs] Target the Proliferating Tumor Cellsand Cancer Stem Cells

Patient derived hGB cells were cultured for 5-7 days in the presence ofthe following NPs:

[1] EGCG—8 μM; [2] curcumin—0.5 μM; [3] Sulforaphane—2.5 μM. Cells werethen dissociated into a single cell suspension and plated at a lowdensity in 96 well plates together with control nutrient growth medium[NeuroCult+EGF]. At this stage cells were no longer exposed to NPs.After being cultured for 7-10 days in the 96 well plates, the number ofspheres was counted [to determine the clonogenic frequency] and thespheres were sized [to determine the proliferative capacity of eachclone forming cell]. Cells in 96-well plates were first fixed with a 4%paraformaldehyde solution containing 0.2% Triton-X and 1:1000 dilutionof DAPI. Fluorescent images were taken and the number of spheres andtheir size quantitated using Macnification. Data was exported to exceland statistical analysis done in GraphPad. FIG. 9 details the results ofthis experiment and demonstrates the ability of our NP combination toreduce the pool of proliferating clone forming precursor cells and toreduce the proliferative ability of the clones. As cancer is a diseasedefined by the uncontrolled growth of clonogenic cancer cells, theability of our NPs to reduce the number of clones and to reduce theproliferative potential of the existing clones points to the novelty andutility of our findings.

In a second experiment [FIG. 10], patient derived hGB cells wereserially passed in culture while being treated with the following NPs:

[1] EGCG—8 μM

[2] Curcumin—0.5 μM

[3] SFN— 2.5 μM

[4] A combination of all three NPs

FIG. 10A demonstrates the ability of each NP on its own to reduce theslope of the growth curve compared to the control cultures. Thecombination of all three NPs produced an unexpected synergistic effect.Applying an algorithm that we have recently developed and published on,the data derived from this serial passage experiment allows us tointerrogate and measure the effect of treatment on the cancer stem cellpopulation. When expressed as a Kll value [the probability of a cancerstem cell undergoing a symmetric cell division over a defined period oftime] we can see that each of our NPs are able to target the expansionof the cancer stem cells. Strikingly, the combination of our three NPshas a statistically significant effect on reducing symmetric cancer stemcell divisions relative to the control or any of the NPs use on theirown [FIG. 10 B].

Together these data demonstrate the ability of our NP combination totarget and reduce the clonogenic population in a fairly aggressive solidtissue tumor and to be able to reduce the proliferative ability of theclones. It is important to note that the reduced proliferation wasobserved when the cells were no longer exposed to the NPs [FIG. 9]. Thiswould suggest that a brief exposure to our NPs might have a lastingeffect on tumor cell proliferation. In addition, the capability of ourNP combination to target the cancer stem cell population is extremelyimportant and relevant as it is this population, which is going to beresponsible for therapeutic resistance and recurrence.

EXAMPLE 6 Natural Products [NPs] Work Synergistically with ConventionalChemotherapy

Standard of care [SOC] for many solid tissue cancers involves the use ofchemotherapy, which for many advanced or high grade tumors providesmarginal benefit. Patient derived hGB cells were grown in culture andtreated daily with the conventional drug temozolomide [TMZ, 20 μM] onlyor in combination with the following NPs:

[1] EGCG—8 μM

[2] Curcumin—0.5 μM

[3] SFN— 2.5 μM

[4] a combination of all three NPs

After 5-7 days in culture cells were counted and the mean daily foldexpansion calculated. While SOC drug TMZ had a statistically significanteffect on reducing the expansion of the tumor cells the addition of EGCGor SFN enhanced this effect, with SFN demonstrating the greatest effect.However, the combination of all three NPs, together with TMZ, had thegreatest effect with there being a statistically significant reductioncompared to all groups. These results demonstrate that our unique NPcombination is capable of enhancing the therapeutic efficacy of SOC TMZby 8 fold.

EXAMPLE 7 The Modified Ketogenic Diet [mKD] is Safe, NutritionallySufficient and as Effective as the Ketogenic Diet [KD] as a CancerTreatment

NOD-SCID animals were placed on one of the following diets:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] KD: 92% Fat, 3% carbohydrate, 5% protein.

[3] mKD: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein.

Body weight was measured 24 days after treatment began and while the KDgroup exhibited a significant decrease in body weight, the mKD treatedanimals maintained their body weight relative to the control dietanimals [FIG. 12]. This supports the notion that the mKD isnutritionally sufficient to support normal health.

Following inoculation of 1M GB tumor cells into the right flank ofNOD-SCID mice animals were randomized and put on one of the three diets.Tumor progression was followed by recording 2 measurements of tumordiameter and converting this into a volume using the following formula:(4/3)πR3. For spheroid tumors the two measurements were averaged todetermine the diameter of the sphere. In the case of ellipsoid tumors(i.e. prolate or oblate spheroid mass) the formula used was:(4/3)π*(d/2)*(d/2)2. In this case the second measurement “d2” wouldcount twice and “d” only once. For prolate spheroids, the longmeasurement occurs once while the short measurement occurs twice.Conversely, for the oblate spheroid tumors, the long measurement occurstwice while the short one occurs only once. Following this criteria,tumor volume was tracked over time. FIG. 13 illustrates that the KD andmKD resulting in a significant delay in tumor progression compared tothe control group. No difference was seen between the KD and mKD fedanimals. Overall survival was analyzed using Kaplan-Meier survival curve[GraphPad], and both the mKD and KD feed animals lived significantlylonger than controls [FIG. 14]. Similar to tumor progression, there wasno significant difference between the mKD and KD groups. Lastly, wecompared the time to tumor progression [defined as the time it took fora palpable tumor to reach a size that was visible—300 mm3], in this casethere were a statistically significant reduction in the time to tumorprogression in both the KD and mKD group [FIG. 15]. No difference wasseen between the KD and mKD feed animals. FIG. 16 depicts the meansurvival of our three treatment groups. Mean survival was significantlyenhanced in both the KD and mKD fed animals, with no differences seenbetween these two groups.

Overall, these data support our conclusion that our mKD is nutritionallysufficient with no adverse effects on overall health and like the KD isan effective cancer treatment that delays tumor progression and enhancesoverall survival.

EXAMPLE 8 Combining the mKD and NPs Enhances the Therapeutic Effect onGB Cell Proliferation and Targeting of the Cancer Stem Cell PopulationIn Vitro

Patient derived hGB cell lines were exposed to either the mKD, NPs orboth for 5-7 days in culture after which total cell number wasdetermined and compared to control cultures. The mKD was mimicked invitro by reducing glucose levels to those found in patients who are on aKD [65-80 mg/dl] and elevating ketones to 4 mM [hydroxybutyrate, Sigma].The three natural products that were added were:

[1] EGCG—8 μM

[2] Curcumin—0.5 μM

[3] SFN—2.5 μM

Under these conditions there was a significant reduction in the numberof cells that were generated over the 5-7 day in both the mKD and NPtreated cultures. The most significant reduction however was seen whenthe mKD was used together with NPs [FIG. 17]. In a separate experimentwe examined the effects of each treatment [mKD, NPs, and combination] onthe number of proliferating or clonogenic cells. Cultured hGB cells weretreated with one of our three treatment conditions for seven days invitro, after which cultures were washed, dissociated into a single cellsuspension and we plated in control medium so as to assess the effectsof treatment on the number of sphere forming cells [or clonogenicfrequency]. The number of spheres was enumerated 7 to 10 days later.FIG. 18 demonstrates that while the mKD and the NPs treatments resultedin an approximate 50% reduction in the number of sphere forming cells[which was not significant due to a high variability] there was astatistically significant reduction in the mKD/NP treated cultures[90%].

Cancer stem cells are thought to contribute to therapy resistance and beresponsible for driving long term tumor growth, targeting thispopulation is widely believed to be essential for the development ofsuccessful cancer therapeutics. Using a previously published algorithmthat is able to enumerate symmetric cancer stem cell divisions, data wascollected by serially passage of patient derived hGB cells in one of ourfour treatment conditions [control, mKD, NP or mKD/NP]. Our dataindicates that each of the treatment conditions resulted in asignificant reduction in the frequency of symmetric cancer stem celldivisions relative to the control. The greatest effect was seen in thecombination treatment of mKD/NP [FIG. 19].

Together the experiments in this example demonstrate that thecombination of a mKD and our NPs exhibit the greatest effect at reducingoverall proliferation, reducing the number of proliferating clonogeniccells and targeting the cancer stem cell population by reducing thenumber of symmetric stem cell divisions within this importantsubpopulation of GB tumor cells.

EXAMPLE 9 Combining mKD and NPs Reduces Tumor Progression, IncreasesOverall Survival Compared to mKD or NPs Alone

NOD-SCID animals were inoculated with 1M patient derived hGB cells. Oncea palpable tumor was identified [2 to 4 weeks later], animals wererandomized into 1 of 4 groups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein.

[3] NP diet: 55% carbohydrate, 30% protein, 15% fat+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

[4] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Tumor were measured 3 times per week, volume calculated and progressiontracked, presented and statistical comparison made in GraphPad [usingnon-linear regression, 2-way ANOVA]. FIG. 20 illustrates tumorprogression for these four treatment groups and demonstrates asignificant reduction in tumor progression for all treatment groupsrelative to control. The combination of mKD/NP demonstrated the mostsignificant reduction compared to any of the treatment groups. Whenanimals reached the endpoint, [tumors greater than 1500 mm3], they werekilled and Kaplan-Meier plot was used to analyze survival [GraphPad].FIG. 21 reveals that all treatment groups survived significantly longerthan the controls, with the mKD/NP treated animals demonstratingstatistically significant increase in survival compared to mKD and NPtreated animals. The mean survival is depicted in FIG. 22 and furtherdemonstrates the superiority of mKD/NP in increasing survival. Thecombination of the two treatments also resulted in a statisticallysignificant increase in progression free survival as determined by thetime it took a palpable tumor to reach a volume of 300 mm3 [FIG. 23].

Together these data demonstrate the advantage of combining two novel andeffective therapeutic protocols [mKD and NPs] to produce an unexpectedsynergistic effect delaying tumor progression and increasing mean andmaximum lifespan.

EXAMPLE 10 The mKD/NP Diet Increases Lifespan in an Orthotopic XenograftModel of GB

NOD-SCID mice received an intracranial injection of 200K hGB cells intothe right striatum. Three days after initial surgery animals wererandomized into one of two groups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Animals were closely monitored for any signs of disease or distress andwhen they began to exhibit abnormal neurological signs [lethargy,paralysis, seizure or abnormal motor behavior] animals were killed.Kaplan-Meier plots indicate that the mKD/NP group survived significantlylonger than the control fed animals [FIG. 24]. The mean survival wascalculated for both groups and FIG. 25 reveals a significant increase inmean survival for the mKD/NP fed animals.

These data strongly support the efficacy of using our combination mKD/NPtreatment in an orthotopic xenograft cancer model.

EXAMPLE 11 mKD/NP Treatment Performs as Well as Standard of CareChemotherapy for GB

The standard of care [SOC] for patients with high-grade gliomas such asGB involves the use of the chemotherapy drug Temozolomide [TMZ]. WhileTMZ demonstrates some degree of effectiveness for GB patients, theincrease in survival is marginal [about 2-3 months] and the negativeside effects are significant [nausea, vomiting and hematologicaltoxicity]. Hence, there exists a need for less toxic but effectivetreatments. NOD-SCID animals were inoculated with 1M patient derived hGBcells. Once a palpable tumor was identified [approximately 2-3 weekspost implant], animals were randomized into 1 of 3 groups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] Standard of Care: Control diet together with daily TMZ injections [5mg/kg].

[3] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Tumors were measured 3 times per week, volume calculated and progressiontracked, presented and statistical comparison made in GraphPad [usingnon-linear regression, two-way ANOVA]. FIG. 26 demonstrates that themKD/NP diet is able to reduce tumor progression to the same extent asSOC. Relative to the control animals both SOC and mKD/NP resulted in asignificant increase in progression free survival as measured by thetime they took a palpable tumor to reach 300 mm3 [FIG. 27].

Together these data indicate that the mKD/NP therapeutic is as effectiveas SOC chemotherapy in delaying tumor progression and enhancingprogression free survival.

EXAMPLE 12 mKD/NP is an Effective Adjunct Treatment when Used Togetherwith Standard of Care for GB

NOD-SCID animals received an injection of 1M patient derived hGB cellsinto the right flank. Once a palpable tumor was identified animals wererandomized into 1 of 4 treatment groups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] Standard of Care: Control diet together with daily TMZ injections [5mg/kg].

[3] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

[4] SOC+mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg), together withdaily TMZ injections [5 mg/kg].

Tumor volume was measured 3 times per week, recorded and analyzed usingGraphPad software. FIG. 28 depicts tumor growth over time and indicatesthat mKD/NP performs as well as SOC; however, the combination of mKD/NPtogether with SOC demonstrates a further reduction in tumor progression.When TMZ-resistant hGB cells were used as the donor cells implanted intothe NOD-SCID mice, SOC had no effect on tumor progression. However,mKD/NP was an effective treatment that was enhanced by combining mKD/NPand SOC [FIG. 29].

In summary, the experiments in this example demonstrate the efficiencyof mKD/NP treatment and that the efficacy can be enhanced by combiningwith SOC. Unexpectedly, SOC resistant tumors are not only responsive tomKD/NP but addition of mKD/NP with TMZ sensitizes the tumor cells toprevious ineffective SOC therapy. As virtually all patients with stageIV cancers will develop resistance to their SOC chemotherapy, theability to sensitize the tumor to SOC therapy with a safe and lowtoxicity adjunct treatment is of tremendous value for the cancer carecommunity.

EXAMPLE 13 mKD/NP as a Preventative Treatment

There are currently nearly 14 million people living with cancer in theUSA alone, a number that is expected to rise to 18M in the next 10years. Developing treatments that prevent or delay initial developmentof cancer or recurrence will have a significant effect on not only thepersonal impact of the cancer but a tremendous economic influence aswell. Towards this end we have tested the ability of the mKD/NPtreatment as a preventive treatment and to delay tumor onset.

NOD-SCID animals were placed on one of two diets:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

After being on the diets for two months animals received a subcutaneousinjection of 1M patient derived hGB cells into the right flank. Animalswere monitored daily for the initial appearance of a palpable tumor[approximately 65 mm3]. FIG. 30 indicates that animals on the mKD/NPdiet had a significant delay in the time to first appearance of a tumor[approximately 300%]. Of greater interest was the 60% reduction in thenumbers of animals that developed tumors [FIG. 31].

Using a similar paradigm, animals were placed on the control or mKD/NPdiet for two weeks prior to being inoculated with 2M lung carcinomacells [A549] in the right flank of NOD-SCID animals. 21 days after tumorimplantation approximately 90% of the control animals had developed apalpable tumor, in contrast, only 50% of the animals on the mKD/NP diethad a palpable tumor [FIG. 32].

Together these data demonstrate the efficacy of the mKD/NP diet in notonly delaying the onset of a discernible tumor but also its ability toreduce the occurrence of tumor and hence have a preventive effect.

EXAMPLE 14 mKD/NP Attenuates the Effects of a Peripheral Tumor onCentral Nervous System Stem Cell Proliferation and NP is Able to EnhanceIn Vitro the Pool of Neural Stem Cells

NOD-SCID animals were inoculated with 1M cells in the right flank. Oncea discernible tumor was palpated, animals were randomized into one oftwo groups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

When tumors reached end point, animals received three injections of BrdU[50 mg/kg] over a 72-hour time period. Animals were killed, brainsremoved, fixed, sectioned and BrdU antibodies used to identify cellsthat were in S-phase during the 72 hour injection period. The number ofBrdU-immunoreactive cells were enumerate in the dentate gyrus of thehippocampus. FIG. 33A reveals that relative to normals [non-tumorbearing animals], peripheral tumors caused a dramatic and significantreduction in the number of proliferating cells. However, in animalstreated with mKD/NP diet there was a striking increase in the number ofproliferating dentate gyrus stem cells. These data indicate that themKD/NP treatment is able to protect the endogenous neural stem cellsfrom the negative effects of a tumor located outside of the CNS. Giventhe growing body of literature demonstrating cognitive impairment incancer patients, this data supports the use of the mKD/NP diet formaintaining normal brain function and memory.

When somatic human neural stem cells (hNSCs) were cultured in vitrousing the Neurosphere Assay and treated with each of the individualnatural products ([1] EGCG—8 μM, 2] Curcumin—0.5 μM or [3] SFN—2.5 μM)or the combination of all three, each natural product on its own did notshow any effect on the viability of hNSCs (analyzed using the standardMTT assay). However, the combination of all three natural productsexhibited a significantly increased viability [FIG. 33B].

Together these data support the notion that mKD/NP and NP representunique combinations able to efficiently enhance and maintain the pool ofsomatic neural stem cells both in vitro and in vivo.

EXAMPLE 15 Optimization of the mKD/NP Diet

To improve the effectiveness of the mKD/NP diet we investigated theaddition of Daikon Radish Sprout Powder [DRSP]. NOD-SCID animals wereinoculated with 1M patient derived GB cells and once a palpable tumorwas identified animals were randomized into 1 or 3 groups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP.001: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg; BSP100%),Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

[3] mKD/NP.002: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Tumor volume was measured three times per week using calipers. FIG. 34demonstrates that mKD/NP.002 had a significant effect on reducing tumorprogression relative to the control [p<0.001] and was statisticallysignificantly better than mKD/NP.001 [p<0.05]. Similarly, the time for apalpable tumor to reach a volume of 300 mm3 was significantly delayed inthe mKD/NP.002 fed animals in comparison to mKD/NP.001 group [FIG. 35].Importantly, mean survival was enhanced in the mKD/NP.002 treatedanimals relative to mKD/NP.001 group [FIG. 36, p<0.05].

Once animals had reached end point the tumors were surgically excisedand dissociated into a single cell suspension so they could be analyzedby using flow cytometry. The cells were fixed [4% PFA] and processed forimmunohistochemistry using an antibody that identified the cellproliferation antigen Ki67 and pSTAT3 (pathway activated in GB that is anumber one driver of uncontrolled proliferation). FIG. 37A illustrates asignificant reduction in the percentage of cells that are activelydividing in the mKD/NP.002 group compared to mKD/NP.001 [p<0.05] andcontrol [p<0.01]. Similarly, there was a marked reduction in thepercentage of GB cells with activated STAT3 signaling as evidenced bythe reduction in the number of pSTAT3 immunoreactive cells [FIG. 37B].

EXAMPLE 16 mKD/NP Treatment is an Effective Therapy for Colon Cancer

Using a colorectal adenocarcinoma cell line [HT-29], 2M cells wereimplanted into the right flank of NOD/SCID animals. Once a palpabletumor was noted, animals were randomly assigned to one of two groups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Tumors were measured 3 times/week and animals sacrificed once the tumorsreach endpoint [1000 mm3]. Tumor volume was measured over time and FIG.38 indicates a significant reduction in tumor progression for the mKD/NPtreated animals. The effects of mKD/NP on delaying tumor progression arefurther reflected in FIG. 39 where tumor volume was compared between thecontrol and the treated group 30 days after tumor inoculation. In thiscase there was a significant reduction in the mean tumor volume[student's t-test, p<0.01].

Importantly, Kaplan-Meier survival curves demonstrated that the mKD/NPtreated animals survive significantly longer than the controls [FIG. 40,p<0.01, Log rank test], which was also reflected in the meantime toreach endpoint [FIG. 41].

When colon cancer cells were treated in vitro with each of theindividual natural products, or the combination of all three, eachnatural product on its own exhibited a significant reduction in thenumber of cells that were generated. However, the combination of allthree natural products demonstrated the largest reduction in cellnumbers.

Together these data support the notion that mKD/NP and NP are able toreduce the proliferation of colon cancer both in vitro and in vivo.

EXAMPLE 17 mKD/NP Treatment is an Effective Therapy for Lung Cancer

Using a lung carcinoma cells (A549), 2M cells were implanted into theright flank of NOD/SCID animals. Once a palpable tumor was noted,animals were randomly assigned to one of two groups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Tumors were measured 3 times/week and animals sacrificed once the tumorsreach endpoint [1000 mm3]. Tumor volume was measured over time and FIG.43 demonstrates a significant reduction in tumor progression for themKD/NP treated animals [p<0.001]. The effects of mKD/NP on delayingtumor progression are further reflected in FIG. 44 where tumor volumewas compared between the control and the treated group 31 days aftertreatment initiation. In this case there was a significant reduction inthe mean tumor volume [student's t-test, p<0.05]. The progression freesurvival of the tumors was determined by measuring the time it tooktumors to go from a palpable stage to a size of 300 mm3. In this casethe progression free survival was significantly delayed in the mKD/NPgroup [FIG. 45]. Importantly, Kaplan-Meier survival curves demonstratedthat the mKD/NP treated animals survive significantly longer than thecontrols [FIG. 46, p<0.05, Log rank test], which was also reflected inthe meantime to reach endpoint [FIG. 47].

When colon cancer cells were treated in vitro with each of theindividual natural products, or the combination of all three, eachnatural product on its own exhibited a significant reduction in thenumber of cells that were generated. However, the combination all threenatural products demonstrated the greatest reduction in cell numbers[FIG. 48].

Together these data support the notion that mKD/NP and NP are able toreduce the proliferation of colon cancer both in vitro and in vivo andthat this unique combination is an effective cancer treatment.

EXAMPLE 18 mKD/NP Treatment is an Effective Therapy for Breast Cancer

Human breast cancer cells [ZR751] grown in culture and treated dailywith physiological concentrations of either EGCG (8 μM), Curcumin (0.5μM) or SFN (2.5 μM) or used in combination (NP). Once the controlcultures had reached confluency, cell numbers were determined using thestandard MTT Assay. FIG. 49 reveals that each of the NPs on their ownhad no statistically significant effect on reducing the number of breastcancer cells, however, the combination of all three NPs reduced thenumber of cells by nearly 40%.

Next 2M ZR751 breast cancer cells were implanted into the flank of aNOD/SCID mouse and host animals randomized into 1 of 2 groups once adetectable tumor could be palpated:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Tumors are measured 3 times per week using calipers and tumor volume iscalculated using the following procedure. Tumor progression was followedby recording 2 measurements of tumor diameter and converting this into avolume using the following formula: (4/3)πR3. For spheroid tumors thetwo measurements were averaged to determine the diameter of the sphere.In the case of ellipsoid tumors (i.e. prolate or oblate spheroid mass)the formula used was: (4/3)π*(d/2)*(d/2)2. In this case the secondmeasurement “d2” would count twice and “d” only once. For prolatespheroids, the long measurement occurs once while the short measurementoccurs twice. Conversely, for the oblate spheroid tumors, the longmeasurement occurs twice while the short one occurs only once. Followingthese criteria, tumor volume was tracked over time. FIG. 50 depicts theprogression of the tumor over time until the animals reached endpoint[1000 mm³] and reveals that the mKD/NP diet results in a significantreduction in overall tumor progression [p<0.0001, two-way ANOVA]. Theability of mKD/NP to effectively reduce tumor progression is alsoreflected in comparing the mean size of the breast tumors at 70 and 145days post treatment initiation. In this case, as depicted in FIGS. 51and 52, there is an approximate 50% reduction in tumor size at day 70[p<0.01, student t-test] and a similar reduction at Day 145,respectively.

Progression free survival was determined by calculating the time it tookthe tumors to grow from a barely palpable stage [approximately 65 mm³]to a tumor of significant size [visually identifiable, 300 mm³]. In thiscase, the mKD/NP diet produced an approximate 20% increase inprogression free survival [FIG. 53, p<0.01, students t-test].Importantly, comparison of the survival curves [Kaplan-Meier] betweenthe two groups indicate that the mKD/NP diet resulted in a statisticallysignificant increase in survival [FIG. 53, p<0.01, log rank test].

Together these data demonstrate the effectiveness of mKD/NP at treatingan aggressive and deadly form of breast cancer.

EXAMPLE 19 Mechanisms of mKD/NP: Cell Death, Cancer Stem Cells and DNADamage

Using patient derived GB cells that were expended in culture, 1M wereimplanted into the right flank of NOD/SCID animals. Once a palpabletumor was identified animals were randomized to one of two treatmentgroups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Tumors were monitored 3 times per week and animals sacrificed when theyreached endpoint [1500 mm³]. Tumors were harvested and prepared [fixedand labeled with DAPI] for identification of the SubG1 population, whichis representative of the apoptotic subpopulation. The percentage ofcells undergoing cell death is significantly increased in the mKD/NPtreated animals [FIG. 55, p<0.001, student t-test].

CD133 is a prospective marker for GB cancer stem cells where an increasein their frequency is indicative of a more aggressive or more difficultto treat tumor. Development of therapies that are able to target thispopulation and reduce the frequency of CD133 positive cells or cancerstem cells are thought to be a critical component in the development ofmore effective therapeutics. Using the paradigm details in this example,we probed the control and mKD/NP treated tumors with a CD133 specificantibody so as to calculate the overall percentage of CD133-positivecancer stem cells. FIG. 56 depicts the results from one particularexperiment where we noted a 60% reduction in the size of theCD133-positive cancer stem cell population. It was also noted, FIG. 57,in the mKD/NP treated animals that the CD133-positive cancer stem cellscontained notable double-stranded DNA breaks approximately 3 times thanthe control treated animals [this was determined using an antibody thatmarks the phosphorylated form of H2AX, which is positively correlated tothe amount of DNA double-stranded breaks].

Together these data demonstrate the ability of mKD/NP to not onlyincrease the incidence of apoptotic cell death but to importantly targetGB cancer stem cells by increasing the amount of DNA damage in thisparticular and clinically relevant subpopulation.

EXAMPLE 20 mKD/NP Targets Known Drivers of Tumor Proliferation andMechanisms that Afford Resistance to Conventional Treatments

Using cell lines that were derived from patient tumors and cultured inserum free media, 1M GB cells were implanted into the right flank ofNOD/SCID animals. Once a palpable tumor was identified animals wererandomized to one of two treatment groups:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

Tumor volume was monitored 3 times per week using calipers and animalswere sacrificed when they reached endpoint [1500 mm³], tumors excisedand cells processed for immunohistochemistry and identification ofintracellular pathway activation. Analysis was performed using flowcytometry. FIG. 58 summarizes the effects of mKD/NP on the expression[FIG. 58A] and activation [FIG. 58B] of Y-box binding protein 1 [YB1], aprotein that is implicated in the maintenance and proliferation of tumorcells [including brain and breast tumors]. The data in FIG. 58demonstrates a significant reduction in overall YB1 expression and inthe number of cells that demonstrate phosphorylation, and henceactivation, of YB1 [FIG. 58 A and B, respectively].

Within this same experimental series we also assessed the CD133+ cancerstem cell population and levels of the anti-apoptotic effector NFkB[FIG. 59] and noted a marked reduction of over 80% in the percentage ofcancer stem cells that were expressing NFkB. This demonstrates theability of mKD/NP to target the anti-apoptotic mechanisms that cancerstem cells use to survive conventional treatments.

In summary, the data from these experiments demonstrate that mKD/NPtargets known drivers of tumor proliferation and anti-apoptoticmechanisms and together provide a better mechanistic understanding ofthe target[s] of mKD/NP.

EXAMPLE 21 mKD/NP Attenuates Chemotherapy Induced Upregulation ofProteins that Contribute to Treatment Resistance

Patient derived GB cells were cultured in the NeuroSphere Assay [usingdefined culture conditions]. The cells were treated daily for 4 of theirseven days in culture with one of the following:

[1] Control

[2] TMZ [10 μM]

[3] NP combination [EGCG (8 μM), Curcumin (0.5 μM) and sulforaphane (2.5μM)]

[4] TMZ [10 μM]& NP combination [EGCG (8 μM), Curcumin (0.5 μM) andsulforaphane (2.5 μM)]

After seven days in culture cells were harvested, fixed with 4% PFA,processed for immunocytochemistry and analyzed by flow cytometry. FIG.60 depicts the increase in MGMT levels in cells that are treated withTMZ [approximately 30%] and the attenuation of this increase [less than10% of the control levels] when the TMZ treated cultures were alsotreated with the NP combination. FIG. 61 depicts the changes in overallsurvivin expression [a member of the inhibitor of apoptosis for whichthe overexpression is associated with chemoresistance] within thepopulation and the number of survivin expressing cells. While TMZresulted in a statistically significant increase in the overallexpression of survivin and in the number of cells that expressed it, theaddition of NP returned the levels back to those that were comparable tocontrol. Interestingly, NP on its own did not affect the expression ofsurvivin compared to the control cells.

Following inoculation of 1M GB cells into the right flank of NON/SCIDanimals, and their randomization into one of two treatment groups,animals were treated with one of two diets:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

When animals reached endpoint they were sacrificed, tumors removed,dissociated into a single cell suspension, fixed in 4% PFA and analyzedby flow cytometry for MGMT or pSTAT3 expression. FIG. 62 summarized theresults of this experiment and teaches that MGMT [FIG. 61A] and pSTAT3[FIG. 61B] levels markedly reduced when treated with mKD/NP relative tocontrols. As both elevated MGMT and pSTAT are mechanism correlated andknown to increase resistance to chemotherapy, mKD/NP demonstrates apromising approach to decrease the expression levels of these proteinsand sensitize cells to chemotherapy.

In summary, the experiments in this example provide a mechanism for ourobserved reduction in tumor progression, increased survival and enhancedresponse to chemotherapy.

EXAMPLE 22 Mechanism for mKD/NP Therapeutic Effect on Reducing TumorProgression and Enhancing Survival

FIGS. 63-66 illustrates a number of mechanisms that mKD/NP diet caninfluence to reduce tumor progression and increase life span. Followinginoculation of 1M GB cells into the right flank of NON/SCID animals, andrandomization into one of two treatment groups, animals were treatedwith one of two diets:

[1] Control diet: Is a standard mouse chow and is composed of 55%carbohydrate, 30% protein, 15% fat.

[2] mKD/NP: 10% carbohydrate, 60% Fat (half coming from medium chaintriglycerides [MCT, Neobee 598]), 30% Protein+SFN (25 mg/kg;BSP95%/DRSP5%), Curcumin (1200 mg/kg), EGCG (1200 mg/kg).

When tumors reached endpoint the animals were sacrificed, tumorsremoved, dissociated into a single cell suspension, fixed in 4% PFA andanalyzed by flow cytometry [FIGS. 63, 64, 65 & 66], immunohistochemisty[FIG. 66B] and western blots [FIG. 66D]. Together, these data illustratethat mKD/NP diet is able to reduce the expression of many of the driversof tumor progression including ZEB1 [FIG. 63], mTOR [FIG. 66], thosethat enhance survival such as NFkB [FIG. 64]. mKD/NP diet is also ableto target a key protein involved in glucose metabolism that isupregulated in GB and that plays a key role in providing glucose, andhence fuel, to actively proliferating GB cells [FIG. 65].

These data support the broad effect that mKD/NP has on tumor cells andits ability to simultaneously target multiple mechanisms that play arole in altering the primary drivers of tumor progression but also theescape mechanisms that are responsible for inherent and acquired tumorresistance.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication. In addition, any elements or limitations of any inventionor embodiment thereof disclosed herein can be combined with any and/orall other elements or limitations (individually or in any combination)or any other invention or embodiment thereof disclosed herein, and allsuch combinations are contemplated with the scope of the inventionwithout limitation thereto.

REFERENCES

-   Deleyrolle, L. P., Harding, A., Cato, K., Siebzehnrubl, F. A.,    Rahman, M., Azari, H., et al. (2011). Evidence for label-retaining    tumour-initiating cells in human glioblastoma. Brain., pp. 1-13.-   Redon, C. E., Dickey, J. S., Nakamura, A. J., Kareva, I. G., Naf,    D., Nowsheen, S., et al. (2010). Tumors induce complex DNA damage in    distant proliferative tissues in vivo. Proceedings of the National    Academy of Sciences of the United States of America, 107(42),    17992-17997.-   Sherry, M. M., Reeves, A., Wu, J. K., & Cochran, B. H. (2009). STAT3    is required for proliferation and maintenance of multipotency in    glioblastoma stem cells. Stem Cells (Dayton, Ohio), 27(10),    2383-2392.-   Fotovati, A., Abu-Ali, S., Wang, P.-S., Deleyrolle, L. P., Lee, C.,    Triscott, J., et al. (2011). YB-1 Bridges Neural Stem Cells and    Brain Tumor-Initiating Cells via Its Roles in Differentiation and    Cell Growth. Cancer Research, 71(16), 5569-5578.-   Gao, Y., Fotovati, A., Lee, C., Wang, M., Cote, G., Guns, E., et al.    (2009). Inhibition of Y-box binding protein-1 slows the growth of    glioblastoma multiforme and sensitizes to temozolomide independent    O6-methylguanine-DNA methyltransferase. Molecular Cancer    Therapeutics, 8(12), 3276-3284.-   Kohsaka, S., Wang, L., Yachi, K., Mahabir, R., Narita, T., Itoh, T.,    et al. (2012). STAT3 inhibition overcomes temozolomide resistance in    glioblastoma by downregulating MGMT expression. Molecular Cancer    Therapeutics, 11(6), 1289-1299.-   Boado, R J., Black, K L., Pardrige, W M. Gene expression of GLUT3    and GLUT1 glucose transporters in human brain tumors. (1994). Gene    expression of GLUT3 and GLUT1 glucose transporters in human brain    tumors. Brain Res Mol Brain Res, 27(1), 51-57.-   Le Calvé, B., Rynkowski, M., Le Mercier, M., Bruyère, C., Lonez, C.,    Gras, T., et al. (2010). Long-term in vitro treatment of human    glioblastoma cells with temozolomide increases resistance in vivo    through up-regulation of GLUT transporter and aldo-keto reductase    enzyme AKR1C expression. Neoplasia (New York, N. Y.), 12(9),    727-739.-   F. A. Siebzehnrubl, D. J. Silver, B. Tugertimur, L. P.    Deleyrolle, D. Siebzehnrubl, M. R. Sarkisian, K. G. Devers, A. T.    Yachnis, M. D. Kupper, D. Neal, N. H. Nabilsi, M. P. Kladde, O.    Suslov, S. Brabletz, T. Brabletz, B. A. Reynolds, D. A. Steindler. A    single pathway linking invasion, chemoresistance and tumor    initiation in glioblastoma. Under review.

1. A method of treating a subject for a proliferative disease,comprising: a) administering to the subject in need of such treatment,an effective amount of a composition comprisingepigallocatechin-3-gallate, a composition comprising curcumin, and acomposition comprising glucosinolates and/or derivatives thereof, suchas glucoraphanin and/or sulforaphane (SFN), and, optionally, providing amodified ketogenic diet or a ketogenic diet to the subject; or b)providing a modified ketogenic diet or a ketogenic diet to the subject;and, optionally, administering to the subject in need of such treatment,an effective amount of a composition comprisingepigallocatechin-3-gallate, a composition comprising curcumin, and acomposition comprising glucosinolates and/or derivatives thereof, suchas glucoraphanin and/or sulforaphane (SFN).
 2. The method of claim 1,wherein the proliferative disease is a cancer.
 3. The method of claim 2,wherein the proliferative disease is a cancer is selected from the groupconsisting of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, AIDS-Related Cancers, AIDS-Related Lymphoma,Anal Cancer, Appendix Cancer, Astrocytoma, Cerebellar Astrocytoma, BasalCell Carcinoma, Bile Duct Cancer, Extrahepatic Bladder Cancer, BladderCancer, Bone Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma,Embryonal Tumors, Cerebral Astrocytoma, Ependymoblastoma,Medulloblastoma, Medulloepithelioma, Pineal Parenchymal Tumors ofIntermediate Differentiation, Supratentorial Primitive NeuroectodermalTumors and Pineoblastoma, Visual Pathway and Hypothalamic cancer, Brainand Spinal Cord Tumors, Breast Cancer, Bronchial Tumors, BurkittLymphoma, Carcinoid Tumor, Gastrointestinal Cancer, Carcinoma of Headand Neck, Central Nervous System Lymphoma, Cervical Cancer, ChronicLymphocytic Leukemia, Chronic Myelogenous Leukemia, ChronicMyeloproliferative Disorders, Colorectal Cancer, Cutaneous T-CellLymphoma, Endometrial Cancer, Ependymoblastoma, Ependymoma, EsophagealCancer, Ewing Family of Tumors, Extracranial Germ Cell Tumor,Extrahepatic Bile Duct Cancer, Eye Cancer, Intraocular Melanoma,Retinoblastoma, Gallbladder Cancer, Gastric (Stomach) Cancer,Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor (GIST),Extracranial Germ Cell Tumor, Germ Cell Tumor, Extragonadal Germ CellTumor, Ovarian Cancer, Gestational Trophoblastic Tumor, Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer,Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Hypopharyngeal Cancer,Intraocular Melanoma Islet Cell Tumors (Endocrine Pancreas), KaposiSarcoma, Kidney (Renal Cell) Cancer, Kidney Cancer, Laryngeal Cancer,Chronic Lymphocytic Leukemia, Chronic Leukemia, Myelogenous Leukemia,Lip and Oral Cavity Cancer, Lung Cancer, Non-Small Cell Lung Cancer,Small Cell Lymphoma, Cutaneous T-Cell Lymphoma, Non-Hodgkin Lymphoma,Macroglobulinemia, Waldenström, Malignant Fibrous Histiocytoma of Boneand Osteosarcoma, Medulloblastoma, Medulloepithelioma, Melanoma,Intraocular Merkel Cell Carcinoma, Mesothelioma, Metastatic SquamousNeck Cancer with Occult Primary, Mouth Cancer, Multiple EndocrineNeoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm, MycosisFungoides, Myelodysplastic Syndromes, Myelodysplastic/MyeloproliferativeDiseases, Myelogenous Leukemia, Multiple, Myeloproliferative Disorders,Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal CancerNeuroblastoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral CavityCancer, Lip and Oropharyngeal Cancer, Osteosarcoma and Malignant FibrousHistiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ CellTumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer,Pancreatic Cancer, Islet Cell Tumors, Papillomatosis, Paranasal Sinusand Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, PharyngealCancer, Pheochromocytoma, Pineal Parenchymal Tumors of IntermediateDifferentiation, Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Pleuropulmonary Blastoma, Primary Central Nervous SystemLymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer,Renal Pelvis and Ureter Cancer, Transitional Cell Cancer, RespiratoryTract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma,Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Ewing Family of TumorsSarcoma, Kaposi Sarcoma, Soft Tissue Sarcoma, Uterine Sezary Syndrome,Skin Cancer (Nonmelanoma), Skin Carcinoma, Merkel Cell, Small Cell LungCancer, Small Intestine Cancer, Squamous Cell Carcinoma, Squamous NeckCancer with Occult Primary Cancer, Supratentorial PrimitiveNeuroectodermal Tumors, T-Cell Lymphoma, Mycosis Fungoides and SezarySyndrome, Testicular Cancer, Throat Cancer, Thymoma and ThymicCarcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvisand Ureter, Gestational Trophoblastic Tumor, Carcinoma of UnknownPrimary Site, Urethral Cancer, Uterine Cancer, Endometrial UterineSarcoma, Vaginal Cancer, Vulvar Cancer, Waldenström Macroglobulinemia,and Wilms Tumor.
 4. The method of claim 2, wherein the cancer isselected from the group consisting of brain cancer, breast cancer, coloncancer, and lung cancer.
 5. The method of claim 1, further comprisingadministering to the subject Daikon radish sprout, a Daikon radishsprout extract or a powder of said extract or the Daikon radish sprout.6. The method of claim 1, wherein the method further comprises anadditional therapy or therapies to treat the proliferative disease. 7.The method of claim 6, wherein the additional therapy or therapies totreat the proliferative disease are selected from radiotherapy,chemotherapy, surgery, small molecule, kinase inhibition, immunotherapy,and/or monoclonal antibody therapy.
 8. The method of claim 7, whereinthe additional therapy or therapies comprise(s) administering one ormore of compound selected from Abiraterone Acetate, Abitrexate(Methotrexate), Abraxane (Paclitaxel Albumin-stabilized NanoparticleFormulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (BrentuximabVedotin), ADE, Adriamycin (Doxorubicin Hydrochloride), Adrucil(Fluorouracil), Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin,Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (PalonosetronHydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil),Aminolevulinic Acid, Anastrozole, Aprepitant, Arimidex (Anastrozole),Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra(Ofatumumab), Asparaginase Erwinia chrysanthemi, Avastin (Bevacizumab),Axitinib, Azacitidine, BEACOPP, Bendamustine Hydrochloride, BEP,Bevacizumab, Bexarotene, Bexxar (Tositumomab and I 131 IodineTositumomab), Bleomycin, Bortezomib, Bosulif (Bosutinib), Bosutinib,Brentuximab Vedotin, Cabazitaxel, Cabozantinib-S-Malate, CAF, Campath(Alemtuzumab), Camptosar (Irinotecan, ydrochloride), Capecitabine,CAPOX, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CeeNU (Lomustine),Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPVBivalent Vaccine), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE,CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex(Clofarabine), Clolar (Clofarabine), CMF, Cometriq(Cabozantinib-S-Malate), COPP, Cosmegen (Dactinomycin), Crizotinib, CVP(COP), Cyclophosphamide, Cyfos (Ifosfamide), Cytarabine, Cytarabine,Liposomal, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide),Dacarbazine, Dacogen, (Decitabine), Dactinomycin, Dasatinib,Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin, iftitox,Denosumab, DepoCyt (Liposomal Cytarabine), DepoFoam (LiposomalCytarabine), Dexrazoxane hydrochloride, Docetaxel, Doxil (DoxorubicinHydrochloride Liposome), Doxorubicin Hydrochloride, DoxorubicinHydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome),DTIC-Dome (Dacarbazine), Efudex (Fluorouracil), Elitek (Rasburicase),Ellence (Epirubicin Hydrochloride), Eloxatin (Oxaliplatin), EltrombopagOlamine, Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride,EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib),Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi),Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet(Doxorubicin Hydrochloride Liposome), Everolimus, Evista (RaloxifeneHydrochloride), Exemestane, Fareston (Toremifene), Faslodex(Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (FludarabinePhosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil),Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI,FOLFIRI-BEVACIZUMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV,Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gefitinib,Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, Gemtuzumab Ozogamicin,Gemzar (Gemcitabine, ydrochloride), Gleevec (Imatinib Mesylate),Glucarpidase, Halaven (Eribulin Mesylate), Herceptin (Trastuzumab), HPVBivalent Vaccine, Recombinant, HPV Quadrivalent Vaccine (Recombinant),Hycamtin (Topotecan Hydrochloride), Ibritumomab Tiuxetan, ICE, Iclusig(Ponatinib Hydrochloride), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum(Ifosfamide), Imatinib Mesylate, Imiquimod, Inlyta (Axitinib),Ipilimumab, Iressa (Gefitinib), Irinotecan I ydrochloride, Istodax(Romidepsin), Ixabepilone, Ixempra (Ixabepilone), Jakafi (RuxolitinibPhosphate), Jevtana (Cabazitaxel), Keoxifene (Raloxifene Hydrochloride),Kepivance (Palifermin), Kyprolis (Carfilzomib), Lapatinib Ditosylate,Lenalidomide, Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil),Leuprolide Acetate, Levulan (Aminolevulinic (Acid), Linfolizin(Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), LiposomalCytarabine, Lomustine, Lupron (Leuprolide Acetate), Lupron Depot(Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), LupronDepot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (LeuprolideAcetate), Marqibo (Vincristine Sulfate Liposome), Matulane (ProcarbazineHydrochloride), Mechlorethamine Hydrochloride, Mesna, Mesnex (Mesna),Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF(Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate),Mitomycin C, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor),Mustargen (Mechlorethamine hydrochloride), Mutamycin (Mitomycin C),Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), NanoparticlePaclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation),Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide),Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilotinib, Nolvadex(Tamoxifen Citrate), Nplate (Romiplostim), Ofatumumab, Omacetaxine,Mepesuccinate, Oncaspar (Pegaspargase), Ontak (Denileukin Diftitox),Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized NanoparticleFormulation, Palifermin, Palonosetron Hydrochloride, Panitumumab,Paraplat (Carboplatin), Paraplatin (Carboplatin), PazopanibHydrochloride, Pegaspargase, Pemetrexed Disodium, Perjeta (Pertuzumab),Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor,Ponatinib Hydrochloride, Pralatrexate, Prednisone, ProcarbazineHydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta(Eltrombopag Olamine), Provenge (Sipuleucel-T), Raloxifenehydrochloride, Rasburicase, R-CHOP, R-CVP, Recombinant HPV BivalentVaccine, Recombinant HPV, Quadrivalent Vaccine, Regorafenib, Revlimid(Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab),Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride),Ruxolitinib Phosphate, Sclerosol Intrapleural Aerosol (Talc),Sipuleucel-T, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V,Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib),Sunitinib Malate, Sutent (Sunitinib Malate), Synovir (Thalidomide),Synribo (Omacetaxine Mepesuccinate), Talc, Tamoxifen Citrate, TarabinePFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin(Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere(Docetaxel), Temodar (Temozolomide), Temozolomide, Temsirolimus,Thalidomide, Thalomid (Thalidomide), Toposar (Etoposide), TopotecanHydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and I 131Iodine Tositumomab, Totect (Dexrazoxane Hydrochloride), Trastuzumab,Treanda (Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide),Tykerb (Lapatinib Ditosylate), Vandetanib, VAMP, Vectibix (Panitumumab),VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar(Vinblastine Sulfate), Vemurafenib, VePesid (Etoposide), Viadur(Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate,Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, VincristineSulfate Liposome, Vinorelbine Tartrate, Vismodegib, Voraxaze(Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride),Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda(Capecitabine), XELOX, Xgeva (Denosumab), Xtandi (Enzalutamide), Yervoy(Ipilimumab), Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib), Zevalin(Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride),Ziv-Aflibercept, Zoledronic Acid, Zolinza (Vorinostat), Zometa(Zoledronic Acid), or Zytiga (Abiraterone Acetate).
 9. The method ofclaim 1, wherein said method comprises providing both a modifiedketogenic diet or a ketogenic diet and an effective amount of acomposition comprising epigallocatechin-3-gallate, a compositioncomprising curcumin, and a composition comprising glucosinolates and/orderivatives thereof, such as glucoraphanin and/or sulforaphane (SFN) tothe subject.
 10. A composition comprising medium chain triglycerides,epigallocatechin-3-gallate, curcumin, and a composition comprisingglucosinolates and/or derivatives thereof, such as glucoraphanin and/orsulforaphane.
 11. The composition of claim 10, further comprising Daikonradish sprout, a Daikon radish sprout extract or a powder of saidextract or the Daikon radish sprout.
 12. The composition of claim 11,wherein the composition is a powder, liquid, emulsion or gel.
 13. Thecomposition of claim 11, wherein said composition comprisingglucosinolates and/or derivatives thereof is provided in the form of apowered cruciferous vegetable, a powdered cruciferous vegetable sprout,a plant of the Brassica and/or powders thereof or sprouts of Brassicaplants and/or powders thereof.
 14. The composition of claim 12, whereinsaid composition comprising glucosinolates and/or derivatives thereof isprovided in the form of a powered cruciferous vegetable, a powderedcruciferous vegetable sprout, a plant of the Brassica and/or powdersthereof or sprouts of Brassica plants and/or powders thereof.
 15. A foodproduct comprising the composition of claim
 11. 16. The food product ofclaim 15, wherein the food product is a liquid food product, a gel or asolid food product.
 17. A food product comprising the composition ofclaim
 12. 18. The food product of claim 17, wherein the food product isa liquid food product, a gel or a solid food product.
 19. A food productcomprising the composition of claim
 13. 20. The food product of claim19, wherein the food product is a liquid food product, a gel or a solidfood product. 21-72. (canceled)